Mesoderm and definitive endoderm cell populations

ABSTRACT

The present invention provides cell populations that are enriched for mesendoderm and mesoderm, and cell populations that are enriched for endoderm. The cell populations of the invention are useful for generating cells for cell replacement therapy. The present invention further provides a method of generating hepatocytes, cell populations enriched for hepatocytes, and a method of hepatocyte replacement therapy.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation of U.S. patent application Ser. No.14/468,211, filed on Aug. 25, 2014, which is a Continuation of U.S.patent application Ser. No. 13/646,995, now U.S. Pat. No. 8,815,591,filed Oct. 8, 2012, which is a Continuation of U.S. patent applicationSer. No. 11/993,432, now U.S. Pat. No. 8,283,168, having a 371(c) filingdate of Jan. 21, 2010, which is the U.S. National Phase of InternationalPatent Application Serial No. PCT/US2006/024462, filed Jun. 23, 2006,which claims priority to U.S. patent application Ser. No. 11/165,930,filed Jun. 24, 2005, now abandoned. The contents of the foregoingapplications are hereby incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos. 2RO 1HL 48834-09 and 2RO 1 HL 65169-02 awarded by the National Institutes ofHealth. The United States government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

During embryonic development, the tissues of the body are formed fromthree major cell populations: ectoderm, mesoderm and definitiveendoderm. These cell populations, also known as primary germ celllayers, are formed through a process known as gastrulation. Followinggastrulation, each primary germ cell layer generates a specific set ofcell populations and tissues. Mesoderm gives rise to blood cells,endothelial cells, cardiac and skeletal muscle, and adipocytes.Definitive endoderm generates liver, pancreas and lung. Ectoderm givesrise to the nervous system, skin and adrenal tissues.

The process of tissue development from these germ cell layers involvesmultiple differentiation steps, reflecting complex molecular changes.With respect to mesoderm and its derivatives, three distinct stages havebeen defined. The first is the induction of mesoderm from cells within astructure known as the epiblast. The newly formed mesoderm, also knownas nascent mesoderm, migrates to different positions that will be sitesof future tissue development in the early embryo. This process, known aspatterning, entails some molecular changes that are likely reflective ofthe initial stages of differentiation towards specific tissues. Thefinal stage, known as specification, involves the generation of distincttissues from the patterned mesodermal subpopulations. Recent studieshave provided evidence which suggests that mesoderm is induced insuccessive waves which represent subpopulations with distinctdevelopmental potential. The mesoderm that is formed first migrates tothe extraembryonic region and gives rise to hematopoietic andendothelial cells, whereas the next population migrates anteriorly inthe developing embryo and contributes to the heart and cranialmesenchyme. These lineage relationships were defined initially throughhistological analysis and have been largely confirmed by cell tracingstudies. While this segregation of developmental fates is well acceptedin the field of developmental biology, to date, there are no availablemethods of isolating mesoderm and endoderm, prior to commitment to theselineages.

The present invention provides a method for isolating mesoderm anddefinitive endoderm cell populations. These cell populations are usefulto identify agents that affect cell growth and differentiation, toidentify genes involved in tissue development, and to generatedifferentiated cells and tissues for cell replacement therapies.

SUMMARY OF THE INVENTION

The present invention provides cell populations that are enriched formesendoderm and mesoderm cells. Mesendoderm cells are defined herein ascells that express brachyury (brach⁺) and which, in the presence ofdifferentiation-inducing conditions, are capable of generating mesodermand mesoderm derivatives including cardiac and skeletal muscle, vascularsmooth muscle, endothelium and hematopoietic cells, and also are capableof generating endoderm and endoderm derivatives including liver cellsand pancreatic cells. Mesoderm cells are defined herein as cells thatare brach⁺ and which, in the presence of differentiation inducingconditions, are capable of generating cardiac and skeletal muscle,vascular smooth muscle, endothelium and hematopoietic cells, and are notcapable of generating endoderm and endoderm derivatives.

The present invention further provides cell populations that areenriched for endoderm cells. Endoderm cells are defined herein as cellsthat do not express brachyury (brach″) and which, in the presence ofdifferentiation-inducing conditions, are capable of generating lungcells, liver cells and pancreatic cells.

The present invention also provides methods of isolating cellpopulations enriched for mesendoderm and mesoderm cells, and cellpopulations enriched for endoderm cells. In another embodiment thepresent invention provides cell populations that are enriched forhepatocytes, and methods of making such populations.

In another embodiment, the invention provides a method of hepatocytereplacement therapy.

In another embodiment, the present invention provides methods ofidentifying agents that affect the proliferation, differentiation orsurvival of the cell populations of the invention. A method ofidentifying genes involved in cell differentiation and development ofspecific lineages and tissues is also provided.

Antibodies that specifically recognize brach cells are also provided.The antibodies are useful, for example, for isolating mesendoderm andmesoderm cell populations.

In another embodiment, the present invention provides a method forgenerating cells in vitro. Such cells are useful, for example, for cellreplacement therapy.

The present invention also provides a transgenic non-human mammal havinga genome in which DNA encoding a selectable marker is present in thebrachyuiy locus such that one brachyury allele is inactivated and theselectable marker is expressed in cells in which the brachyury locus istranscribed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the scheme of the vector and the strategy used fortargeting the green fluorescence protein (GFP) to the brachyury locus.

FIGS. 2A and 2B depict the expression of GFP and brachyury in developingembryoid bodies (EBs). FIG. 2A depicts the kinetics of brachyuryexpression determined by reverse transcriptase-polymerase chain reaction(RT-PCR). FIG. 2B depicts the kinetics of GFP expression determined byfluorescence activated cell sorting (FACS) analysis. Numbers above thefigure in FIG. 2A and the histograms in FIG. 2B represent day of EBdifferentiation.

FIGS. 3A, 3B and 3C depict the developmental potential of wild type andGFP-Bry ES cells. FIG. 3A is a histogram showing developmental potentialof day 6 EBs. (Mac/Ery: colonies of macrophages and definitive erythroidcells; Mac: pure macrophage colonies; Ery^(d): colonies of definitiveerythroid cells; Mix: multilineage colonies; Ery^(p): primitiveerythroid colonies. FIG. 3B is a histogram depicting blastcolony-forming cell (BL-CFC) potential of EBs. FIG. 3C shows geneexpression patterns during EB development for wild-type and GFP-Brycells. Numbers at the top of the lanes represent day of EBdifferentiation.

FIGS. 4A and 4B depict the gene expression profile of EB fractionsisolated on the basis of GFP. FIG. 4A shows the profile of GFPexpression in day 3.5 EBs. 1 and 2 represent the gates used to isolatethe GFP⁻ and GFP⁺ fractions. FIG. 4B depicts RT-PCR expression analysisof isolated fractions.

FIGS. 5A, 5B and 5C demonstrate the isolation and characterization ofGFP and Flk-1 populations. FIG. 5A depicts the profiles and gates usedto isolate the GFP⁻/Flk-1⁻, GFP⁺/Flk-1⁻ and GFP⁺/Flk-1⁺ fractions fromday 3.0 and 3.5 EBs Numbers next to the gates represent the threedifferent populations. FIG. 5B shows the Blast colony (Blast) andsecondary EB (2°) potential of the different fractions. FIG. 5C showsthe expression analysis of the isolated fractions. Expression shown inthe top panel was evaluated using a polyA⁺ global amplification PCRmethod described by Brady et al. (1990) Meth. In Mol. And Cell Bio.2:17-25. The data in the lower panels was obtained by RT-PCR analysisusing gene specific oligonucleotides. Numbers on the top of each rowindicate the cell population as designated in FIG. 5A.

FIG. 6 depicts the expression of GFP and Flk-1 in isolated day 3EB-derived fractions. The top row shows the expression profiles of thethree fractions prior to culture (pre). The bottom row indicates theprofile of the same cell populations following 20 hours of culture(post). The numbers below each profile indicate the BL-CFC and primitiveerythroid (Ery^(P)-CFC) potential (precursors per 1×10⁵ cells plated) ofeach population.

FIGS. 7A and 7B depict the BL-CFC potential and Flk-1 expression of theisolated cell populations prior to and following culture. In FIG. 7A,the numbers on the bottom refer to the cell population: 1 is thepresort, 3 is the GFP⁺/Flk-1⁻ fraction and 4 is the GFP⁺/Flk-1⁺fraction. Cells were cultured for 20 hours, and the aggregates were thendissociated and analyzed for BL-CFC. Data are shown for cells isolatedfrom day 3, 3.5 and 4.0 EBs. In FIG. 7B, the top row representsGFP⁺/Flk-1⁻ cells isolated from day 3.0, 3.5 and 4.0 EBs prior toculture (pre). The bottom row shows the Flk-1 expression pattern of thesame fraction, following culture (post). Numbers above the barsrepresent the percentage of Flk-1⁺ cells.

FIGS. 8A and 8B demonstrate the effects of BMP-4 and fetal calf serum(FCS) on the development of brachyury and Flk-1⁺ in/on day 3.0 EBderived cells under the conditions indicated at the top of eachhistogram. FIG. 8B depicts expression of brachyury and Flk-1 on cellpopulations generated from GFP+/Flk-1⁻ cells cultured for 20 hours underthe indicated conditions.

FIG. 9 is a schematic model of mesoderm formation and specification inEBs.

FIG. 10 shows the expression of genes in EBs in the presence and absenceof serum.

FIG. 11 is a graph depicting brachyury expression in EBs generated underdifferent conditions.

FIG. 12 shows that EBs grown in serum contained hematopoieticprecursors, whereas those grown without serum did not.

FIG. 13 is a schematic diagram showing neuronal differentiation is thepresence and absence of serum.

FIG. 14 shows the expression of genes in EBs initiated for two days inserum and then switched to serum free conditions.

FIG. 15 shows gene expression in EBs cultured in the presence of bFGF.

FIG. 16 shows gene expression patterns in Bry⁺ and Bry⁻ cells culturedin the presence of bFGF.

FIG. 17 is a diagram of the mesoderm and endoderm populations of thepresent invention and the differentiation of these population toderivative cell types.

FIG. 18 depicts the kinetics of expression of GFP (brachyury) and Flk-1in EBs differentiated for 2.5, 3.0, 3.5 and 4.0 days. Arrows indicatethe GFP⁺ population isolated used for the analyses in subsequentstudies.

FIG. 19 depicts the hemangioblast and cardiac potential of the GFP⁺populations isolated from the four stages of EB differentiation. Cellsfrom each stage were isolated by cell sorting, reaggregated for 24 hoursand analyzed for hematopoietic and cardiac potential. Data are indicatedas blast colonies (hemangioblast) per 1×10⁵ cells recovered from thereaggregation culture or as the % of aggregates that gave rise tobeating cell masses indicative of cardiac muscle differentiation.

FIG. 20 provides the RT-PCR expression analysis of the indicated genesin the four GFP⁺ EB-derived cells populations. Numbers indicate day ofEB differentiation.

FIG. 21 shows HNF3 β expression in GFP⁺ populations isolated from day3.0 and 4.0 EBs. Pre represents cells prior to sorting, −/− are cellsthat express no GFP or Flk-1 and +/− represents the GFP⁺ Flk-1⁻population.

FIGS. 22A, 22B and 22C demonstrate the effects of activin on developmentof EBs in serum-free cultures. A) FACS profile showing GFP expression inday 6 EBs differentiated in the presence of 100 ng/ml of activin. B)Kinetics of GFP induction in cultures containing 100 ng/ml of activin.Open circles are EB differentiated in the presence of activin, closedsquares are EBs differentiated in absence of activin. C) RT-PCRexpression analysis of indicated genes in day 6 EBs grown in thepresence (+activin) or absence (−activin) of activin. Numbers indicateday of EB differentiation.

FIGS. 23A and 23B show the effects of different concentrations ofactivin on the developmental potential of EBs. A) GFP expression in day7 EBs induced with different concentrations of activin. B) RT-PCRexpression analysis of day 7 EBs induced with different concentrationsof activin.

FIGS. 24A and 24B show the hematopoietic progenitor content of EBsdifferentiated in the presence of different concentrations of activin.A) Progenitor potential of day 7 EBs, Ep are primitive erythroidprogenitors, mac/mix represent definitive hematopoietic progenitors. B)Progenitor potential of day 7 activin-induced EBs following 2.5 days ofexposure to serum.

FIG. 25 shows the development of albumin expressing cells from GFP⁺cells induced with ether 3 or 100 ng/ml of activin. GFP⁺ and GFP⁻ cellswere isolated at day 6 of differentiation and cultured for a further 8days in the conditions previously described to support hepatocytedifferentiation.

FIGS. 26A and 26B depict three-week old renal grafts of bry⁺ (FIG. 26A)and bry⁻ (FIG. 26B) cell populations. FIG. 26C depicts sections ofgrafts of the bry⁺ and bry⁻ populations.

FIG. 27A is a FACS profile indicating the bry⁺/c-kit⁺ (+/+) andbry⁺/c-kit⁻ (+/−) fractions isolated from day three serum-stimulatedEBs. Numbers represent the proportion of cells in each of the fractions.FIG. 27B shows expression analysis of each of the fractions. Day 3represents cells analyzed immediately following sorting. Day 15represents cell populations cultured for 15 days in hepatocyteconditions.

FIG. 28 depicts expression analysis of cell populations derived from EBsinduced with different concentrations of activin. Numbers at the top ofthe figure indicate activin concentration. Numbers at the bottom of thefigure represent an estimate of the proportion of EBs with skeletalmuscle outgrowths.

FIG. 29 depicts expression analysis of brachyury fractions isolated fromactivin-induced populations. Numbers at the top of the figure indicateactivin concentration.

FIGS. 30A and 30B depict the method used to target human CD4 (hCD4) intothe HNF3β locus. FIG. 30A depicts the scheme of the vector and strategyused for targeting CD4. FIG. 30B shows the southern blot of ES cellclones electroporated with the targeting vector.

FIGS. 31A, 31B and 31C depict the expression of GFP, hCD4, Brachyury,and HNF3β in developing EBs induced with serum. FIG. 31 A shows dotplots of GFP versus hCD4 expression determined by flow cytometricanalysis. FIG. 31B shows the mean fluorescence intensity (MFI) of bothGFP and hCD4 expression of the plots in part A. The MFI is displayed asa percent of maximum detected during the time course for each marker.FIG. 31C depicts the expression of Brachyury and HNF3β by RT PCR in thesame samples used in part A for flow cytometry.

FIGS. 32A, 32B and 32C depict gene expression in different GFP hCD4expressing population as well as tissue dissected from the primitivestreak of mouse embryos. EBs were differentiated in serum for 3.25 daysand sorted by the expression of GFP and hCD4. FIG. 32A shows dot plotsof GFP versus hCD4 expression before and after cell sorting for thesepopulations. GFP⁺ hCD4^(lo), GFP⁺ hCD4^(med), and GFP+ hCD4^(hi)populations were sorted to the purity indicated. FIG. 32B showsexpression of genes by RT PCR in the samples sorted in part A. FIG. 32Cshows expression of genes by RT PCR in tissue dissected from theprimitive streak dissected from day 7.25 embryos. Abbreviations used:Pre, presort; ant, anterior primitive streak tissue; mid, middleprimitive streak tissue, pos, posterior primitive steak tissue.

FIGS. 33A, 33B and 33C depict the developmental potential of differentGFP hCD4 expressing populations. EBs were differentiaited and cellsorted as in FIG. 32. Only two populations were sorted, GFP⁺ hCD4^(lo)and GFP⁺ hCD4^(hi) cells. After sorting, cells were reaggregated inserum replacement containing medium for 2 days. FIG. 33A shows cardiacpotential of the sorted populations after plating on matrigel another24-48 hours. The percentage of EBs containing beating cells wascalculated. FIG. 33B shows hematopoietic potential of the sortedpopulations determined by plating in methylcelluose with hematopoieticcytokines. FIG. 33C shows expression of endodermal genes by RT PCR ofthe sorted populations after plating on matrigel for an additional 5days in serum free media.

FIGS. 34A, 34B and 34C depict ES cells differentiated in serum freeconditions with Activin A Wnt3a and/or inhibitors of these pathways. EScells were cultured in serum free media for two days then trypsinized toobtain a single cell population. These cells were then reaggregated withthe indicated conditions. At day six, the EBs were plated on matrigelcoated dishes in serum free media. FIG. 34A shows dot plots of GFPversus hCD4 expression for days 3 through 6 with EBs that werereaggregated with media alone, Activin A 25 ng/ml, Wnt3a 100 ng/ml, orthese stimulations plus either DKK1 lOOng/ml or SB-431542 6 uM. FIG. 34Bshows RT PCR gene expression data from RNA obtained from cultures shownin FIG. 34A. FIG. 34C shows RT PCR gene expression data from RNAobtained from EBs plated on matrigel for an addition 6 days that hadinitially been stimulated with media alone, Activin or Wnt3a asindicated above.

FIGS. 35A and 35B depict, the effects of DKK1 and activin on EBdifferentiation. EBs differentiated and sorted as in FIG. 32 for GFP⁺hCD4^(med) were allowed to reaggregate in serum replacement containingmedia alone or with the addition of human DKK1 100 ng/ml, activin 100ng/ml, or both factors together. FIG. 35A shows histograms for hCD4 andGFP expression of the sorted cells after 1 day reaggregation. FIG. 35Bdepicts endodermal gene expression by RT PCR. The sorted populationswere treated as indicated above for 2 days, then plated on matrigel inserum free media without factors added. Gene expression was analyzed anadditional 5 days after plating.

FIGS. 36A, 36B and 36C shows flow cytometry analysis (36A, B) and geneexpression analysis (36C) of hepatic colonies.

DETAILED DESCRIPTION OF THE INVENTION

During embryogenesis, the formation of mesoderm is a critical step inthe establishment of a body plan and in the development of multipleorgan systems such as blood, endothelium, heart and skeletal muscle. Themolecular mechanisms that control mesoderm formation, however, arepoorly defined. A model system based upon the differentiation ofembryonic stem (ES) cells in culture has been used to studymesodermal-derived populations including hematopoietic, endothelial,cardiac and skeletal muscle and adipocyte lineages. The in vitro modelsupports the induction and specification of mesoderm, but thesedifferentiation events take place in complex colonies known as embryoidbodies (EBs) generated from ES cells. It would be advantageous toisolate mesoderm cell populations from EBs as they are formed, in orderto better understand mesoderm formation and tissue development. However,it has not been possible to isolate these populations by cell sortingusing antibodies, because antibodies specific for nascent mesoderm cellpopulations are not well-defined.

Brachyury (also known as T) is the founding member of a family oftranscription factors known as T-box genes and was first identified as anaturally occurring mutation in mice. Papaioannou et al. (1998)Bioessays 20:9-19. Heterozygous mice are viable but have a shorter tailthan wild type animals. Homozygous mutants, which die at approximatelyday 10 p.c, lack a notochord and display defects in the development ofposterior mesodermal tissues. Through the analysis of chimeric animals,brachyury has been shown to affect the migratory properties of themesodermal cells. Wilson et al. (1995) Development 121:877-86.Expression analysis revealed a unique and interesting pattern forbrachyury. It is expressed transiently in all cells ingressing throughthe primitive streak as well as in the nascent and early migratingmesoderm. Wilkinson et al. (1990) Nature 343:657-9; Herrmann et al.(1991) Development 113:913-7. Expression is rapidly downregulated inparaxial, lateral and extraembryonic mesoderm and following regressionof the steak, is confined to the tailbud and notochord. Given thispattern, brachyury is considered to be one of the best markers of earlymesoderm and is used to track the development of this lineage. Brachyuryhas been identified in all species analyzed, suggesting that its role inmesoderm development is preserved throughout phylogeny. Papaioannou etal. (1998).

In accordance with the present invention, a selectable marker gene hasbeen recombinantly targeted to the brachyury locus. It has beendiscovered that, following the initiation of ES cell differentiation,the selectable marker is expressed in a pattern that reflects brachyuryexpression. The selectable marker has allowed the sorting of brachyurypositive (Brach⁺) cells from EBs, and thereby the isolation andcharacterization of cell populations that are enriched for mesendodermand mesoderm cells.

The selectable marker exemplified in accordance with the presentinvention is the enhanced green fluorescence protein (EGFP or GFP).Other selectable markers that will facilitate cell sorting are known tothose of ordinary skill in the art and may be used in the presentinvention. The cDNA encoding GFP is known in the art (and iscommercially available, for example as plasmid pEGFP.Cl from Clontech,Palo Alto, Calif.), and may be targeted to the brachyury locus byconstructing targeting vectors (GFP-Bry) by methods known in the art.The vectors are preferably designed to replace approximately two-thirdsof the first exon of the brachyury gene with a GFP expression cassette.

Brachyury genes from numerous species, including human and mouse, areknown in the art and reviewed, for example, by Smith (1997) CurrentOpinion in Genetics & Development 7:474-480. The GFP expression cassettepreferably contains GFP cDNA and one or more translational stop codonsto prevent translation of downstream brachyury exons. The cassette mayfurther contain an exon encoding the SV40 polyadenylation signalsequence to prevent transcription of downstream regions of the brachyurygene.

The vectors are introduced into ES cells by methods known in the art tointegrate the GFP-Bry construct by homologous recombination. ES cellsmay be isolated from blastocysts by methods known in the art anddisclosed for example by Evans et al. (1981) Nature 292:154-156, Thomsonet al. (1995) Proc. Nat'l. Acad. Sci. USA 92; 7844; U.S. Pat. No.5,843,780; and Reubinoff et al. (2000) Nature Biotech. 18:399. In apreferred embodiment the ES cells are mouse or human ES cells. Followingsuccessful targeting the brachyury start codon becomes the start codonof GFP, resulting in the disruption of the targeted brachyury allele.The resulting cells are designated GFP-Bry ES cells. GFP-Bry ES cellsare defined herein as ES cells in which one brachyury allele isinactivated and GFP is expressed under the control of the brachyuryregulatory elements.

It has been discovered in accordance with the present invention thatGFP-Bry ES cells, in which one brachyury allele is inactivated, areviable and develop and differentiate normally. Further, it has beendiscovered that GFP expression mirrors endogenous brachyury expression.Accordingly, brach⁺ cells may be isolated by selecting for cells thatexpress GFP. Cells that express GFP may conveniently be isolated by flowcytometry, for example by fluorescence-activated cell sorting (FACS).Methods for sorting cells based on fluorescent properties are well-knownto those of ordinary skill in the art.

The present invention also provides GFP-Bry ES cells that have aselectable marker gene recombinantly targeted to the HNF3β locus. HNF3βis known to be expressed in most endodermal cell types. HNF3β is alsoknown as Foxa2, and these terms are used interchangeably herein. Themarker allows the sorting of HNF3β+ cells. The marker exemplified inaccordance with the present invention is a truncated human CD4 thatlacks most of the intracellular domain. This cell surface molecule ispreferred because it cannot transduce signals and antibodies areavailable for flow cytometry. Other markers that will facilitate cellsorting are known to those of ordinary skill in the art and may be usedin the present invention. The cDNA encoding human DC4 is known in theart and may be targeted to the HNF3β locus by methods known in the art.A targeting vector having cDNA encoding the truncated human CD4 clonedbetween two arms of homology of HNF3β may be electroporated into GFP-BryES cells to provide CD4-HNF/GFP-Bry ES cells. The expression of humanCD4 in these cells reflects endogenous expression of HNF3β.

Cell populations that are enriched for mesendoderm and mesoderm cells,as defined hereinabove, may be obtained by culturing GFP-Bry ES cells inthe presence of serum for a time sufficient to obtain GFP⁺ cells, forexample for from about one to about four days for mouse cells, andsorting and isolating GFP⁺ cells, for example by flow cytometry. Thecell population that is isolated contains at least about 50%, andpreferably at least about 75%, and more preferably at least about 90%,and most preferably at least about 95% or at least about 99% mesendodermand mesoderm cells. The relative amounts of mesendoderm and mesoderm maybe varied by adjusting the length of the culture in serum, with shorterculture times favoring the presence of mesendoderm and mesodermpatterned to the hematopoietic and endothelial lineages, and longerculture times favoring the presence of mesoderm patterned to the cardiacand skeletal muscle lineages. For example, a cell population enrichedfor mesoderm may be obtained by culturing in serum for about 2.5 to 4.5days, followed by sorting and isolating GFP⁺ cells. Culturing in thepresence of serum is defined herein as culturing in media supplementedwith animal serum, for example fetal calf serum (FCS). In a preferredembodiment, the media is supplemented with from about 5% to about 25%serum. The optimal concentration may be serum batch dependent and can bedetermined by one of ordinary skill in the art.

Cell populations that are enriched for mesendoderm and mesoderm cellsmay be obtained from GFP-Bry ES cells generated from human ES cells by asimilar method in which the length of time of culture in serum islengthened to account for difference in times of differentiation invitro for human and mouse cells. Accordingly, GFP-Bry ES cells generatedfrom human ES cells are cultured in serum for a time sufficient toobtain GFP⁺ cells, for example about 2 to about 18 days, before sortingand isolating GFP⁺ cells.

For both mouse and human cell populations, it can be easily determinedwhether the isolated cells have differentiated beyond mesoderm, forexample to hemangioblasts, by assaying for the presence of the tyrosinekinase receptor, human KDR or mouse. KDR and are not expressed inmesendoderm and nascent mesoderm, but as these cells differentiate to ahemangioblast/pre-erythroid population, KDR or expression is detectable.KDR⁺ and Jflk-1⁺ cells may be identified by flow, cytometry usingantibodies to KDR or Flk-I. Such antibodies are known in the art, andmay also be generated using standard methods of antibody production. Thecell populations enriched for mesendoderm and mesoderm may be furtherenriched by removing KDR or Flk-I⁺ cells by cell sorting.

As depicted in FIG. 17, it has been discovered in accordance with thepresent invention that mesendoderm is a previously unidentified cellpopulation that gives rise to both endoderm and mesoderm and theircorresponding lineages. It has been further discovered that presence orabsence of serum in the in vitro culture may be used to dictate whichlineage is generated from mesendoderm. In particular, a cell populationthat is enriched for endoderm cells may be obtained by culturing GFP-BryES cells generated from mouse ES cells in the presence of serum forabout two to four days, sorting and isolating GFP⁺ cells, for example byflow cytometry, followed by culturing the GFP in the absence of serumfor from about one to about ten days. The cell population that isisolated contains at least 50%, and preferably at least about 75%, andmore preferably at least about 90%, and most preferably at least about95% or at least about 99% endoderm cells, as defined hereinabove.

Cell populations that are enriched for endoderm cells may be obtainedfrom GFP-Bry ES cells generated from human ES cells by culturing theGFP-Bry ES cells in the presence of serum for about 2 to 10 days, andthen sorting and isolating GFP⁺ cells followed by culturing the GFP⁺cells in the absence of serum for from about 1 to about 15 days.

The populations enriched for endoderm cells may be further enriched byidentifying and sorting out KDR⁺ or Flk-I⁺ cells as described above.

It has further been discovered in accordance with the present inventionthat cell populations enriched for endoderm may be obtained by culturingGFP-Bry embryonic stem cells in the absence of serum and in the presenceof the growth factor activin or nodal, for about two to about ten days,and isolating cells that express brachyury. The amount of activin ornodal is sufficient to induce differentiation of embryonic stem cells toendoderm. Such differentiation may be measured by assaying for theexpression of genes associated with endoderm development, including forexample HNF3β, Mix1-1, Sox17, Hex-1 or pdx-1. In a preferred embodiment,the concentration of activin is at least about 30 ng/ml. In anotherpreferred embodiment the concentration of activin is about 100 ng/ml. Inanother embodiment, the GFP-Bry embryonic stem cells are cultured in theabsence of serum and the presence of activin or nodal and an inhibitorof Wnt signalling for about two to about 10 days, and cells expressingbrachyury are isolated. In a preferred embodiment the inhibitor isDKK-I. In other preferred embodiments the concentration of DKK-I is atleast about 30 ng/ml, or about 100 ng/ml.

Cell populations enriched for mesoderm may be obtained by culturingGFP-Bry embryonic stem cells in the absence of serum and the presence ofactivin or nodal for about two to about ten days, and isolating cellsthat express brachyury. The amount of activin or nodal is sufficient toinduce differentiation of embryonic stem cells to mesoderm, butinsufficient to induce differentiation to endoderm.

Differentiation to mesoderm may be measured by assaying for theexpression of genes associated with mesoderm development, including forexample GATA-I, and the absence of expression of genes associated withendoderm development. In a preferred embodiment, the concentration ofactivin is less than 30 ng/ml. In another preferred embodiment theconcentration of activin is about 3 ng/ml.

It has been further discovered in accordance with the present inventionthat cell populations enriched for endoderm may be obtained by culturingembryonic stem cells in the absence of serum and the presence of activinor the related protein nodal and a Wnt molecule for about one to aboutsix days, isolating brach⁺ HFN3β3 cells, and culturing the isolatedcells in the absence of serum and the presence of an inhibitor of Wntsignaling for at least about one day. Isolation of brach⁺/HFN3β3⁺ cellsmay be facilitated by using the C4-HNF/GFP-Bry ES cells describedhereinabove and selecting for CD4 and GFP, for example by cell sorting.

Wnt refers to a family of polypeptides well-known in the art. See, e.g.U.S. Pat. No. 6,159,462. The Wnt growth factor family includes proteinsencoded by at least ten genes in mice and at least seven genes in human.In a preferred embodiment of the present invention, the Wnt molecule isrecombinant Wnt 3d.

Inhibitors of Wnt signalling are also well-known in the art and include,for example, Wnt-I disclosed in U.S. Pat. No. 6,844,422 and Dickkopf-1(DKK-I) disclosed by Glinka et al. (1998) Nature 391:357-362.

The present invention also provides a method of making cell populationsenriched for endoderm comprising culturing mouse embryonic stem cells inthe absence of serum for about two to about four days, or humanembryonic cells in the absence of serum for about two to about ten days,isolating brach⁺/HFN3β3⁺ cells, and culturing the isolated cells forabout one to about ten days in the absence of serum and the presence ofactivin or nodal and an inhibitor of Wnt signalling. Isolation ofbrach⁺/HFN3β3⁺ cells may be facilitated by using the CD4-HNF/GFP-Bry EScells as described above.

In each of the foregoing two methods, the amounts of activin or nodaland Wnt inhibitor are sufficient to induce differentiation of ES cellsto endoderm. Differentiation may be measured by assaying for theexpression of genes associated with endoderm development. In a preferredembodiment the concentration of activin is at least about 30 ng/nil. Inanother preferred embodiment the concentration of activin is about 100ng/ml. In a preferred embodiment the Wnt inhibitor is DKK-I and theconcentration of DKK-I is at least about 30 ng/ml. In another preferredembodiment the concentration of DKK-I is about 100 ng/ml.

The present invention further provides a method for making cellpopulations enriched for hepatocytes comprising culturing mouseembryonic stem cells in the absence of serum and the presence of activinor nodal for about two to four days, or human embryonic cells in theabsence of serum and the presence of activin or nodal for about two toten days, isolating brach⁺/HFN3β3⁺/cKit⁺ cells, culturing the isolatedcells in the absence of serum and the presence of BMP-4 and bFGF underconditions sufficient for the production of hepatoblasts, and culturingthe hepatoblast under conditions sufficient for the maturation of thehepatoblasts to hepatocytes. The amount of activin or nodal issufficient to induce differentiation of ES cells to endoderm. In apreferred embodiment, activin is present at a concentration of at leastabout 30 ng/ml, BMP-4 is present at a concentration of at least about 10ng/ml, and bFGF is present at a concentration of at least about 1.0ng/ml. Hepatocytes may be identified by assays for glycogen storage orby observation, for example by electron microscopy, of characteristicsof hepatocytes such as a large nucleus/cytoplasmic ratio, a large amountof glycogen storage and the presence of bile canaliculi.

The foregoing method provides cell populations that comprise at leastabout 10% hepatocytes. In a preferred embodiment, the cell populationscomprise at least about 50% hepatocytes. In more preferred embodiments,the cell populations comprise at least about 60%, or at least about 70%,or at least about 80%, or most preferably at least about 90%hepatocytes.

The present invention further provides a method of identifying agentsthat affect the proliferation, differentiation or survival of the cellpopulations described above. The method comprises culturing cells fromone of the cell populations described hereinabove in the absence andpresence of an agent to be tested, and determining whether the agent hasan effect on proliferation, differentiation or survival of the cellpopulation. The agent to be tested may be natural or synthetic, onecompound or a mixture, a small molecule or polymer includingpolypeptides, polysaccharides, polynucleotides and the like, an antibodyor fragment thereof, a compound from a library of natural or syntheticcompounds, a compound obtained from rational drug design, or any agentthe effect of which on the cell population may be assessed using assaysknown in the art, for example standard proliferation and differentiationassays as described in U.S. Pat. No. 6,110,739. Such agents are usefulfor the control of cell growth and differentiation in vivo and in vitro.

The present invention further provides a method of identifying genesinvolved in cell differentiation and development of specific lineagesand tissues. The method comprises isolating populations Of GFP⁺ cells ofthe invention after different amounts of time in culture, comparing geneexpression profiles in the different populations, and identifying genesthat are uniquely expressed in a population. In a preferred embodiment,microarray analysis and subtractive hybridization are used to comparegene expression profiles.

In another embodiment, the present invention provides methods of makingantibodies that recognize brachyury positive (brach⁺) cells but notbrachyury negative (brach″) cells. Polyclonal antibodies may be made byinjecting an animal with the cells of the invention in an immunogenicform. Also, antibodies may be made by identifying cells surface markerspresent in GFP⁺ but not GFP″ cells, and making antibodies against themarkers or fragments thereof. The antibodies may be monoclonal orpolyclonal, and may be fragments, genetically engineered antibodies,single chain antibodies, and so on. Antibodies may be made by methodswell-known in the art. Such antibodies are useful for identifying andisolating brach cells such as mesendodeπn and mesoderm.

The present invention also provides a method for generating mammaliancells in vitro. In one embodiment, the method comprises culturing cellsfrom a cell population enriched in mesendoderm and mesoderm cells underconditions effective for the differentiation of mesoderm into cardiacmuscle, vascular smooth muscle, endothelium or hematopoietic cells.Conditions effective for differentiation into the various cell types invitro are known in the art. In another embodiment, the method comprisesculturing cells from a cell population enriched in endoderm cells underconditions effective for the differentiation of endoderm into livercells or pancreatic cells. Effective conditions for such differentiationare known in the art. The production of insulin-producing pancreaticislet cells is specifically contemplated.

As demonstrated in accordance with the present invention, brach⁺ cellsisolated from different aged EBs have different developmentalpotentials. BrachVFlk″ cells from about day 3 mouse EBs efficientlygenerate hemotopoietic and endothelial lineages, while the cells fromabout day 3 to 10 EBs generate cells of cardiomyocyte lineages.Accordingly, by adjusting the time of culture of the ES cells used forobtaining the cell population enriched for mesendoderm and mesoderm, oneof ordinary skill in the art can select for efficient production ofhemotopoetic and endothelial lineages or cardiomyocyte lineages.

Such cells are useful, for example, for cell replacement therapy for thetreatment of disorders that result from destruction or dysfunction of alimited number of cell types. Such disorders include diabetes mellitus,liver failure, heart failure, cardiovascular and other vascular disease,Duchenne's muscular dystrophy, osteogenesis imperfecta, and disorderstreatable by bone marrow transplant, for example leukemias and anemias.See, Odorico et al., (2001) Stem Cells 19:193-204.

The cell populations of the present invention are useful for generatingdifferentiated cells and tissues for cell replacement therapies. Thesuitability of the cell populations of the present invention for cellreplacement therapy may be assessed by transplanting the cells intoanimal models of disorders that are associated with the destruction ordysfunction of a limited number of cell types. For example, thefumarylacetoacetate (FAH) deficient mouse disclosed for example byGrompe et al. (1993) Genes & Dev. 7:2298, incorporated herein byreference, provides a model for liver failure. FAH deficient mice sufferfrom progressive liver failure and renal tubular damage unless treatedwith NTBC (2-(2-nitro-4-trifluoromethyl benzoyl)-1,3-cyclohexedione) ortransplanted with normal hepatocytes. These mice thus provide an idealmodel for testing the potential of cells with characteristics ofimmature hepatocytes generated from EBs. Methods for transplantation ofhepatocytes into FAH deficient mice removed from NTBC are known in theart and disclosed for example by Oversturf et al. (1996) Nature Genet.12:266-273. Normal liver function is indicated by survival of the- mice,and may also be assessed by measuring serum aspartate transaminaselevels, plasma bilirubin levels, and by determining normal structure ofthe regenerated liver.

Animal models for other disorders that result from the destruction ordysfunction of particular cells types are known in the art. Such modelsmay similarly be used to assess other cell populations of the presentinvention.

In a preferred embodiment, the present invention provides a method ofhepatocyte replacement therapy comprising administering to a subject inneed of such treatment a composition comprising hepatocytes produced bythe method of the invention. In a preferred embodiment the subject is ahuman. The composition may be administered by a route that results indelivery to hepatic tissue, including, for example, injection,implantation or infusion. The present method is useful for the treatmentof liver failure, liver-based metabolic disease, and chronic liverdisease.

The present invention also provides a transgenic non-human mammal inwhich DNA encoding a selectable marker is present in the brachyury locussuch that one brachyury allele is inactivated and the selectable markeris expressed in cells in which the brachyury locus is transcribed. In apreferred embodiment the mammal is a mouse and the selectable marker isGFP. In particular, the transgenic mouse has a genome comprising atransgene in which a DNA sequence encoding GFP is operably linked tobrachyury regulatory elements, and the transgene is expressed in cellsthat normally express brachyury. The transgenic mouse may be obtained byinjecting the GFP-Bry ES cells described hereinabove into blastocysts,which are then implanted into pseudopregnant females. Transgenic pupsare identified by the short-tail phenotype associated with brach +/−,and by molecular analysis. Such transgenic animals are useful forobtaining early embryos from which to isolate mesoderm to be used inaccordance with the methods of the invention, and for theidentification, isolation and characterization of any adult cellpopulations that express the brachyury gene. Such cells may representnovel stem cell populations.

All references cited herein are incorporated herein in their entirety.

The following examples serve to further illustrate the presentinvention.

Example 1 Materials and Methods

ES Cell Growth and Differentiation.

ES cells were maintained on irradiated embryonic feeder cells inDulbecco's Modified Eagle Medium (DMEM) supplemented with 15% fetal calfserum (FCS), penicillin, streptomycin, LIF (1% conditioned medium) and1.5×10⁻⁴ M monothioglycerol (MTG; Sigma). Two days prior to the onset ofdifferentiation, cells were transferred on gelatinized plates in thesame media. For the generation of EBs, ES cells were trypsinized andplated at various densities in differentiation cultures. Differentiationof EBs was carried out in 60 mm petri grade dishes in IMDM supplementedwith 15% FCS, 2 mM L-glutamine (Gibco/BRL), transferrin (200 ug/ml), 0.5mM ascorbic acid (Sigma), and 4.5×10⁻⁴ M MTG. Cultures were maintainedin a humidified chamber in a 5% CO₂/air mixture at 37° C.

Serum Free Medium.

Two different serum-free media were used in different aspects of thefollowing examples: IIMD supplemented with Knockout™ SR (Gibco BRL) andStemPro® 34 (Gibco BRL).

Methylcellulose Colony Assay. A) Blast Colonies:

For the generation of blast cell colonies (BL-CFC assay), EB-derivedcells were plated at 0.5×-1.5×10⁵ cells/ml in 1% methylcellulosesupplemented with 10% FCS (Hyclone), vascular endothelial growth factor(VEGF; 5 ng/ml), c-kit ligand (KL; 1% conditioned medium), IL-6 (5ng/ml) and 25% D4T endothelial cell conditioned medium (Kennedy et al.(1997) Nature 386:488-93). Transitional colonies were generated in theabsence of VEGF. Colonies were scored following four days of culture. B)Hematopoietic colonies: For the growth of primitive and definitivehematopoietic colonies, cells were plated in 1% methylcellulosecontaining 10% plasma-derived serum (PDS; Antech), 5% protein-freehybridoma medium (PFHM-II; Gibco-BRL) plus the following cytokines:c-kit ligand (KL; 1% conditioned medium), erythropoietin (2 U/ml), IL-11(25 ng/ml), IL-3 (1% conditioned medium), GM-CSF (3 ng/ml), G-CSF (30ng/ml), M-CSF (5 ng/ml), IL-6 (5 ng/ml) and thrombopoietin (TPO; 5ng/ml). Cultures were maintained at 37° C., 5% CO₂. Primitive erythroidcolonies were scored at day 5-6 of culture, whereas definitive erythroid(BFU-E), macrophage, and multilineage colonies were counted at 7-10 daysof culture. C-kit ligand was derived from media conditioned by CHO cellstransfected with KL expression vector (kindly provided by GeneticsInstitute). IL-3 was obtained from medium conditioned by X63 AG8-653myeloma cells transfected with a vector expressing IL-3. VEGF, GM-CSF,M-CSF, IL-6, IL-11, activin BMP2, BMP4, bFGF, FGF8, and lhh werepurchased from R&D systems.

Reaggregation Cultures.

Cells were cultured at 2×10⁵ per ml IMDM supplemented with 15% FCS (orKnockout SR), 2 mM L-glutamine (Gibco/BRL), 0.5 mM ascorbic acid(Sigma), and 4.5×10⁻⁴ M MTG in 24-well petri-grade plates. These wereused to prevent adhesion of the cells to the bottom of the well.

Cardiac Muscle Assays.

GFP⁺ cells were reaggregated in IMDM supplemented with 15% serumreplacement. Twenty hours later the aggregates were cultured in wells ofeither a 24- or 96-well plate in IMDM with 10% serum replacement(serum-free). The wells were pre-treated with gelatin. Cultured weremonitored daily for the development of the appearance of beating cells.Beating cells were usually detected between days 2 and 6 of culture.

Cell Surface Markers Staining and FACS Analysis.

Standard conditions were used to stain the cells. Stained suspensionswere analyzed on a FACScan™ (Becton Dickinson, CA).

Gene Expression Analysis

For the poly A⁺ RT-PCR analysis the method of Brady et al. ((1990) Meth.in Mol. and Cell Bio. 2:17-25) was used. Reverse transcription, poly-Atailing and PCR procedures were performed as described, with theexception that the X-dT oligonucleotide was shortened to5′-GTTAACTCGAGAATTC(T)₂₄-3′ (SEQ. ID. NO. 1). The amplified productsfrom the PCR reaction were separated on agarose gels and transferred toa Zeta-Probe® GT membrane (Biorad) or transferred to the membrane with aslot blot apparatus (Schleicher & Schuell). The resulting blots werehybridized with ³²P randomly primed cDNA fragments (Ready-to-GoLabelling, Pharmacia) corresponding to the 3′ region of the genes (forall except β-H1). A β-H1-specific probe was prepared by annealing twooligonucleotides, (5′-TGGAGTCAAAGAGGGCATCATAGACACATGGG-3′ (SEQ. ID. NO.2), 5′-CAGTACACTGGCAATCCCATGTG-3′ (SEQ. ID. NO. 3)) which share an 8base homology at their 3′ termini. This β-H1 specific oligonucleotidewas labeled with ³²P using a Klenow fill-in reaction. For gene specificPCR, total RNA was extracted from each sample with RNeasy® mini kit andtreated with RNase free DNase (Qiagen). Two microgram of total RNA wasreverse-transcribed into cDNA with random hexamer using a Omniscript™ RTkit (Qiagen). PCR was carried out using appropriate oligonucleotides.The PCR reactions were performed with 2.5 U of Taq polymerase (Promega),PCR buffer, 2.5 mM MgCl₂, 0.2 uM of each primer and 0.2 mM dNTP. Cyclingconditions were as follows; 94° C. for 5 min followed by 35 cycles ofamplification (94° C. denaturation for 1 min, 60° C. annealing for 1min, 72° C. elongation for 1 min) with a final incubation at 72° C. for7 min.

Example 2 Generation of Targeted ES Cells

Under appropriate conditions in culture, embryonic stem (ES) cells willdifferentiate and form three dimensional colonies known as embryoidbodies (EBs) that contain developing cell populations from a broadspectrum of lineages. Smith (2001) Annu. Rev. Cell Dev. Biol. 17:435-62.Among these EB-derived populations, one can detect mesodermalderivatives including those of the hematopoietic, endothelial, cardiacmuscle and skeletal muscle lineages.

In order to track the onset of mesoderm in EBs and to isolate cellsrepresenting this population, the green fluorescence protein (GFP) wastargeted to the brachyury locus. The targeting construct contained theGFP cDNA, and artificial intron, SV40 poly(A) sequences and a loxPflanked neo cassette in the first exon and is depicted in FIG. 1. Thethymidine kinase (TK) gene was included at the 3′ end of the targetingconstruct to select against random integration. The targeting vector wasconstructed as follows.

A BAC clone carrying the entire mouse Brachyury (Bry) gene was isolatedby PCR screening of a 129/Ola strain genomic library (Genome Systems)with primers 5′-AAGGAGCTAACT AACGAGATGAT-3′ (SEQ. ID. NO. 4) and5′-TACCTTCAGCACCGGGAACAT3′ (SEQ. ID. NO. 5). These primers anneal withinthe first and second Bry exon, respectively, and amplify a diagnosticband of ˜600 bp. An approximately 3 kb long PstI restriction fragmentcarrying the 1 exon of the Bry gene along with more than 2 kb of 5′flanking region was identified and subcloned from the BAC into plasmidpBSK (Strategene), resulting in construct pBSK.Bry-5′. Approximately 2kb of the region immediately upstream of the start codon were sequencedto identify appropriate primer annealing sites for the construction ofvectors.

Oligos 5′-GCTAGCTAATGGATCCA-3′ (SEQ. ID. NO.6)/5′-GATCTGGATCCATTAGCTAGCTGCA-3′ (SEQ. ID. NO. 7) and5′-GATCTTAATGAACGGCAGGTGGGTGCGCGTCCGGA G-3′ (SEQ. ID. NO.8)/5′TCGACTCCGGACGCGCACCCACCTGCCGTTCATTAA-3′ (SEQ. ID. NO. 9) wereinserted into the PstI/SalI sites of plasmid pBSK to create a new, moresuitable polylinker with two successive translational stop codons and anartificial 3′ splice site (construct pBry-AA). Plasmid pEGFP.Cl(Clontech) was double-digested with NheI/Bglll and the resulting ˜760 bpDNA fragment encoding EGFP without stop codon was cloned into theNheI/BglII sites of pBRY-AA, resulting in construct pBry-AB. AnXhoI/SalI fragment of plasmid pL2-Neo2 carrying a loxP-flanked neomycinresistance gene was inserted into the SalI site of pBry-AB to give riseto plasmid pBry-AC (transcription of EGFP and Neo in same direction).

A 556 bp XmaI/MluI fragment carrying a consensus splice donor site, anartificial intron, a splice acceptor site and a short exon including theSV40 polyadenylation sequence, was excised from the commercialexpression vector pBK-CMV (Stratagene). This fragment was inserted intoplasmid pBry-AC in the following way: the XmaI end was ligated into theBspEI site following the last EGFP codon, whereas the Mlu end wasinserted along with oligos 5′-CGCGTTACTAGTAAGACGTCT-3′ (SEQ. ID. NO.10)/5′-CCGGAGA CGTCTTACTAGTAA-3′ (SEQ. ID. NO. 11) as linkers into theBspEI site located just upstream of the loxP-neo-loxP cassette.Resulting construct: pBry-AE. An ˜1.9 kb XhoI/SalI fragment encoding theHSV thymidine kinase gene was inserted into the XhoI site of pBry-AE toallow selection against random integration (construct pBry-AH). ANotI/Eco47Ill fragment encoding the “short arm” of homology was excisedfrom pBry-AF and cloned into the NotI/Eco47llI sites of pBry-AH to giverise to plasmid pBry-AI. The “long arm” of homology was excised frompBry-AK with SalI and inserted in the correct orientation into the Sailsite of pBry-AI to yield final targeting vector B.

Embryonic stem cells (E14. 1, 129/Ola Hooper et al. (1987) Nature326:292) were electroporated with NotI-linearized targeting vectorpBry-AM. Four dishes with transfected cells were subjected to G418single- and another four dishes to G418+Gancyclovir (Ganc)double-selection. Clones that had undergone a homologous recombinationevent were identified by PCR with one primer (5′-CAGGTAGAACCCACAACTCCGAC-3′ (SEQ. ID. NO. 12)) annealing to genomic sequences in the 5′region of the Bry gene, just upstream of the “short arm of homology”,the other (5′-CCGGACACGCTGAACTTGTGGC-3′ (SEQ. ID. NO. 13)) to the 5′portion of EGFP (diagnostic band: approximately 1.3 kb). Correctlytargeted clones were confirmed by Southern blot analysis: genomic DNA ofcandidate clones was digested with HincII and hybridized to a probelocated outside of the targeting construct. The probe was derived fromthe Bry 5′ flanking region (−2018 to −1249 with respect to the Bry ATGstart codon) by PCR using the oligonucleotide pair5′-ACAGGATCCCTAAGCCTCAAAAGAGTCGCT-3′ (SEQ. ID. NO.14)/5′-TCTTGGATCCTCCTAT CCTATCCCGAAGCTCCT-3′ (SEQ. ID. NO. 15). 384 G418single- and 80 G418+Ganc double-selected clones were screened, of which4 respectively 3 proved to be positive, corresponding to a targetingefficiency of 1.04% and 3.75%. Two positive clones were transientlytransfected with a modified Cre recombinase expression vector to removethe neo gene. These targeted clones are referred to hereinafter asGFP-Bry ES cells.

Brachyury is expressed transiently in developing EBs with the onsetpreceding the expression of genes that define the establishment of thehematopoietic and endothelial lineages. To determine whether GFPexpression in GFP-Bry ES cells accurately reflects expression of thebrachyury gene during EB development, GFP expression was assessed.

A typical expression pattern during a 6-day EB differentiation period isshown in FIG. 2A. In this experiment, low levels of message weredetected within 24 hours of differentiation. Expression was upregulatedover the next 48 hours, persisted through day 4 and then declinedsharply to undetectable levels by day 6 of differentiation. GFPexpression, as defined by FACS analysis, followed a similar temporalpattern. Low levels of GFP⁺ cells (˜5%) were detected as early as day 2of differentiation. More than half (65%) of the EB-derived cellsexpressed GFP at day 3 and almost all the cells were positive at day 4of differentiation. As observed by PCR, expression dropped sharply afterthis point and by day 6 few, if any, GFP⁺ cells were present. This rapiddecline in GFP expression indicated that it did not persist within thecells for extended periods of time. The high proportion of GFP⁺ cells atdays 3 and 4 of differentiation suggests that development of mesodermwithin the EBs under these conditions is extensive. Taken together,these findings indicate that GFP expression accurately reflectsexpression of the brachyury gene during EB development.

The possibility that inactivation of a single brachyury allele couldhave detrimental effects on the in vitro developmental potential of theES cells was assessed. As indicated, heterozygous mice demonstrate amild phenotype. To determine if the GFP-Bry ES cells display anydetectable defects in hematopoietic development, EBs generated from themwere analyzed for hematopoietic precursor and blast colony-forming cell(BL-CFC) content and for gene expression patterns. The data in FIGS. 3Aand 3B indicate that GFP-Bry ES cells generate comparable numbers ofprimitive (Ery^(p)) and definitive (Ery^(d), Mac, Mac/Ery, and Mix)hematopoietic precursors and BL-CFC compared to the wild type cells.Gene expression patterns (FIG. 3C) confirmed the precursor analysis andshow little difference between the EBs generated from the GFP-Bry andwild type ES cells. Both sets of EBs showed a decline in Rex-1expression over the first 3 days of differentiation. Rex-1 is atranscription factor that is expressed in ES cells and downregulated asthey undergo differentiation. Rogers et al. (1991) Development113:815-24. The decline in Rex-1 is followed by the typical transientwave of brachyury expression which immediately precedes the onset ofgenes involved in the development of the hematopoietic and endotheliallineages. Flk-1, a receptor tyrosine kinase essential for theestablishment of these lineages (Shalaby et al. (1995) Nature 376:62-6)is expressed between day 3 and 6 of differentiation. GATA-1, ahematopoietic transcription factor, and the embryonic and adult globingenes, βH1 and βmajor, were detected at low levels at day 4 ofdifferentiation. Expression of all 3 genes was upregulated over the next24 hours, reflecting the expansion and maturation of the primitiveerythroid lineage at this developmental stage. Palis et al. (1999)Development 126:5073-84. The precursor numbers and the gene expressionpatterns observed in this example are consistent with those found inprevious studies and indicate that the molecular programs leading to theestablishment of the hematopoietic system are intact in the targetedGFP-Bry ES cells.

Example 3 Isolation of Brachyury⁺ Cells

To determine if brachyury⁺ cells could be isolated based on GFPexpression, the GFP⁺ population from day 3.5 EBs was sorted and analyzedfor expression of appropriate genes. FIG. 4A shows the gates used forthe isolation of the GFP positive (2) and negative (1) populations.RT-PCR analysis revealed brachyury expression was restricted to the GFP⁺fraction indicating that cell sorting based on GFP expression can beused to isolate brachyury⁺ cells. Flk-1, one of the earliest makers ofhematopoietic and endothelial development, was present at higher levelsin GFP⁺ that in the GFP⁻ fraction indicating that it was expressed in atleast a subpopulation of brachyury⁺ cells. In contrast to brachyury andFlk-1, Pax-6, a gene involved in early neuronal development [79,80], wasexpressed at higher levels in the GFP⁻ fraction consistent withprecursors of this lineage being brachyury negative. These cell-sortingstudies indicate that expression of GFP under the control of thebrachyury locus provides a novel marker for the isolation,characterization and manipulation of brachyury⁺ cells from EBs.

This example demonstrates that GFP⁺ cells can be isolated from day 3.5EBs by cell sorting. Gene expression analysis of the GFP⁺ and GFP⁻fractions shows that brachyury expression segregates predominantly tothe positive fraction, a finding which clearly demonstrates thatfractionation based on GFP provides a method for isolating brachyurypositive cells. In addition to brachyury, the receptor tyrosine Flk-1involved in early hematopoietic and endothelial development is alsoexpressed at higher levels in the positive than in the negativefraction. In contrast, Rex-1 and Pax-6, markers of early ectoderm andneuroectoderm, are expressed in the GFP⁻ fraction. These findingsdemonstrate that expression of GFP in the context of brachyury can beused to separate mesoderm from ectoderm.

Example 4 Separation of Brachyury Positive Cells into SubpopulationsBased Upon Flk-1 Expression

Flk-1 has been shown to be essential for the establishment of thehematopoietic and endothelial lineages in the early embryo and isexpressed on the earliest precursors of these lineages, including theBL-CFC [Faloon et al. (2000) Development 127:1931-41]. Given thispivotal role in blood and vascular development, its expression withinthe GFP⁺ population was hypothesized to define a subpopulation ofmesoderm undergoing specification to these lineages. To investigate thispossibility further, EBs were analyzed at several stages of developmentfor the presence of GFP and Flk-1 positive cells. In the experimentillustrated in FIG. 5A, 4.8% of the day 3.0 EB population expressed GFPbut not Flk-1, whereas 1.2% of the cells expressed both markers. Thesize of both fractions increased dramatically over the next 12 hourswith the GFP⁺/Flk-1⁻ and GFP⁺/Flk-1⁺ cells representing 52% and 26% ofthe total EB population, respectively. To assess the developmentalpotential of the three populations defined by GFP and Flk-1 expression,GFP⁻/Flk-1⁻ (fraction 2), GFP⁺/Flk-1⁻ (fraction 3) and GFP⁺/Flk-1⁺(fraction 4) cells were isolated at both time points and analyzed forBL-CFC and 2° EB content and for gene expression patterns. The potentialof the fractions was compared to that of the pre-sorted population(fraction 1). The majority of the BL-CFC were found in the GFP⁺/Flk-1⁺fraction at both day 3.0 and 3.5 of differentiation (FIG. 5B). This isnot surprising given that previous studies have shown that all BL-CFCexpress Flk-1. The 2° EB were restricted to the GFP⁻/Flk-1⁻ fraction, afinding consistent with the fact that they derive from residualundifferentiated ES cells in the primary EBs. The GFP⁺/Flk-1⁻ fractiongenerated few colonies under the conditions used in these cultures. Thegene expression analysis revealed some interesting differences betweenthe populations isolated at the 2 time points (FIG. 5C). Rex-1, the EScell marker, was expressed at lower levels in the GFP⁺ than in the GFP⁻fraction, indicating that these populations are undergoingdifferentiation. Brachyury was expressed in the GFP⁺ fractions at bothtime points. The levels appear to be higher in the GFP⁺/Flk-1⁻ than theGFP⁺/Flk-1⁺ fraction isolated from day 3.5 EBs, suggesting that itsexpression could be downregulated as these cells mature towards thehematopoietic and endothelial lineages. As expected, Flk-1 was expressedpredominantly in the GFP⁺/Flk-1⁺ cells at both time points. Scl, ahelix-loop-helix transcription factor that is essential for bothprimitive and definitive hematopoietic development (Shivdasani et al.(1995) Nature 373:432-4), appears to be restricted to the GFP⁺/Flk-1⁺fraction. Similarly, the transcription factor Runx1, required forestablishment of the definitive hematopoietic system (Wang et al. (1996)Proc. Natl. Acad. Sci. 93:3444-9), was most readily detected inGFP⁺/Flk-1⁺ fraction. There was some Runx1 expression in the GFP⁺/Flk-1⁻fraction isolated from day 3.0 EBs. Nodal is expressed in all 3fractions at day 3 of differentiation. At day 3.5, the levels ofexpression in the GFP⁺/Flk-1⁺ fraction appear to be significantly lowerthan in the other fractions. Wnt3a and Wnt8a showed a remarkablyrestricted pattern of expression and were found only in the GFP⁺/Flk-1⁻fraction at both time points, consistent with an early mesoderm functionprior to the expression of lineage restricted markers. BMP2 wasexpressed in both GFP⁺ fractions whereas BMP4 was found predominantly inthe GFP⁺/Flk-1⁺ cells, indicating that these molecules play a role atdistinct stages of development in this system. The BL-CFC potential andgene expression pattern of the GFP⁺/Flk-1⁺ cells indicates that they arerepresentative of the extraembryonic mesoderm found in the mouse embryo.

This example demonstrates that the brachyury fraction of day 3 and day3.5 EBs can be separated into two fractions based on Flk-1 expression:brachyury⁺/Flk-1⁻ (GFP⁺/Flk-1⁻) and brachyury⁺/Flk-1⁺ (GFP⁺/Flk-1⁺)(FIG. 5A). Functional studies demonstrated that precursors (BL-CFC) ableto generate both hematopoietic and endothelial cell segregated to the(GFP⁺/Flk-1⁺) fraction, suggesting that upregulation of Flk-1 isindicative of commitment to these lineages (FIG. 5B). Gene expressionstudies demonstrated distinct differences between the GFP⁺/Flk-1⁻ andGFP⁺/Flk-1⁺ populations (FIG. 5C).

Example 5 Developmental Relationships Among the GFP/FLK Fractions

The expression patterns observed in FIG. 5 are consistent with theinterpretation that the three fractions represent a developmentalcontinuum with the GFP⁻/Flk-1⁻ cells giving rise to the GFP⁺/Flk-1⁻cells which in turn give rise to the GFP⁺/Flk-1⁺ cells. To determine ifthese fractions do represent specific stages within a commondevelopmental pathway, each was isolated from day 3 EBs, cultured for 20hours and then re-analyzed for GFP and Flk-1 expression. BL-CFC andEry^(p)-CFC potential was determined for each of the populations priorto and following culture. The isolated cells were cultured at densitiesof 1×10⁵ cells or greater in petri-grade 24-well plates in the samemedium used for EB differentiation. Under these conditions, the cellsrapidly reaggregate and form EB-like structures that appear to follow anormal developmental program with little expansion or loss in cellnumber. Following the 20-hour reaggregation culture, the GFP⁻/Flk-1⁻cells gave rise to a significant population of GFP⁺/Flk-1⁻ cells as wellas to a small number of GFP⁺/Flk-1⁺. GFP⁺/Flk-1⁻ cells generated asubstantial population of GFP⁺/Flk-1⁺ cells during the same cultureperiod. The GFP⁺/Flk-1⁺ population appeared to lose some GFP and Flk-1expression following the reaggregation culture. Results are shown inFIG. 6. The changes in precursor potential were consistent with thechanges in surface markers. The GFP⁻/Flk-1⁻ fraction, the most immatureof the three, contained an undetectable number of BL-CFC and Ery^(p)-CFCbefore or after culture. The GFP⁺/Flk-1⁻ fraction also contained fewBL-CFC and Ery^(p)-CFC prior to culture. However, following culture, theBL-CFC potential increased dramatically, from 74 to 1564 per 10⁵ cells,consistent with the increase in Flk-1 expression. The frequency ofEry^(p)-CFC did not change during the culture period. The GFP⁺/Flk-1⁺fraction contained BL-CFC but few Ery^(p)-CFC prior to culture. NoBL-CFC were detected following culture, however, the population nowcontained Ery^(p)-CFC. Together with the surface marker analysis, theseprecursor data support a developmental progression from a pre-mesoderm(GFP⁻/Flk-1⁻) to a mesoderm/pre-hemangioblast (BL-CFC) population(GFP⁺/Flk-1⁻) to a hemangioblast/pre-erythroid population (GFP⁺/Flk-1⁺)to a post hemangioblast/erythroid population (possiblyGFP^(lo)/Flk-1^(lo)). Not all the cells in a given population appear todifferentiate following the 20-hour culture period as cells with thestarting phenotype persisted in the GFP⁻/Flk-1⁻ and GFP⁺/Flk-1⁻cultures.

This example indicates that when isolated and recultured for 20-24hours, each of the three populations isolated from day 3 EBs continuedto differentiate and in a pattern that indicates that these populationsrepresent a developmental continuum. For instance, GFP⁻/Flk-1⁻ gave riseto GFP⁺/Flk-1⁻ cells which in turn gave rise to GFP⁺/Flk-1⁺. Thesechanges in cell surface characteristics were associated with theexpected changes in developmental potential. The GFP⁺/Flk-1⁻ fractioncontained few hematopoietic/endothelial precursors (BL-CFC) prior toculture. Following culture, these precursors were detected, clearlydemonstrating that the GFP⁺/Flk-1⁻ fraction from day 3 EBs does containthe potential to give rise to Flk-1⁺ cells with hematopoietic andendothelial potential.

Example 6 Potential of GFP/Flk-1⁻ Cells

The foregoing examples demonstrated that GFP⁺/Flk-1⁻ cells isolated fromday 3.0 EBs efficiently generated GFP⁺/Flk-1⁺ cells and BL-CFC followingovernight culture. To determine if this pre-BL-CFC potential wasspecific to the GFP⁺/Flk-1⁻ fraction isolated at this stage ofdevelopment, GFP⁺/Flk-1⁻ cells from different aged EBs were assayed fortheir ability to give rise to BL-CFC. As shown in FIG. 7A, the capacityto generate BL-CFC was most robust in the day 3 GFP⁺/Flk-1⁻ cells. Thisdevelopmental potential decreased dramatically by day 3.5 ofdifferentiation and was almost non-existent at day 4.0. The BL-CFCcontent of the freshly isolated GFP⁺/Flk-1⁺ fraction from these same EBsincreased over this period of time, indicating that differentiation wasprogressing in a normal fashion. The Flk-1 expression patterns in thereaggregated cultures from the different staged EB cells were consistentwith BL-CFC data. The cultures from the reaggregated day 3.0 GFP⁺/Flk-1⁻cells contained a distinct Flk-1 fraction that represented more than 40%of the total population (FIG. 7B). Flk-1 expression in the day 3.5 and4.0 cultures was significantly lower and consisted of a shoulder of thetotal population rather than a distinct peak.

Example 7 Cardiomyocyte Potential of GFP and Flk-1 Subpopulations

Given the sequence of events in the mouse embryo in which thedevelopment of hematopoietic and endothelial mesoderm is followed by thedevelopment of cardiac and cranial mesoderm, the cardiomyocyte potentialof the isolated populations was determined. For this analysis,aggregates from the cultures of the different populations weretransferred to microtiter or 24-well plates in serum-free medium andmonitored for the development of beating cell masses, indicative ofcardiomyocytes. These conditions are known to support the efficientdevelopment of cardiomyocytes from the reaggregated cells. As anindependent confirmation of the cardiomyocyte nature of the cells withinthese masses, a representative group was transferred to microscopeslides, fixed and stained for the presence of the cardiac-specificisoform of Troponin T. All beating cell masses analyzed were found tocontain Troponin T positive cells indicating that they werecardiomyocytes. Using this assay, the cardiomyocyte potential of thereaggregated GFP⁺/Flk-1⁻ and GFP⁺/Flk-1⁺ fractions from different stagedEBs was determined. For comparison, the BL-CFC potential of the freshlyisolated GFP⁺/Flk-1⁺ cells and of the cultured GFP⁺/Flk-1⁻ cells wasanalyzed.

TABLE I BL-CFC, pre-BL-CFC and cardiomyocyte potential of theGFP+/Flk-1− and GFP+/Flk-1+ fractions isolated from different stagedEBs. GFP⁺ Flk⁻ GFP⁺ FIk⁺ BL-CFC beating BL-CFC beating EB followingfollowing direct following Age culture culture plating culture 2.75 +++− + − 3.5 + ++ +++ − 4.0 +/− ++ +++ −

As shown in Table 1, the BL-CFC potential of the different fractions wassimilar to that observed in previous experiments. The GFP⁺/Flk-1⁺ cellsisolated from the three different EB populations generated BL-CFC, withthe highest number found at day 3.5 and 4.0. The pre-BL-CFC potential inthe GFP⁺/Flk-1⁻ cells was greatest at day 2.75 and decreasedsignificantly at 3.5 and 4.0. The cardiomyocyte potential of thefractions showed a reverse pattern. A significant proportion (>80%) ofthe transferred aggregates from day 3.5 and 4.0 GFP⁺/Flk-1⁻ cellsgenerated beating cardiomyocytes. No beating cells were observed in theaggregates generated from the earliest (day 2.75) GFP⁺/Flk-1⁻ cells.Beating cells were not detected in aggregates generated from theGFP⁺/Flk-1⁺ cells isolated from EBs at any of the three stages ofdevelopment. The findings from this analysis are consistent with thenotion that GFP⁺/Flk-1⁻ cells isolated at different stages havedifferent potentials. Those that develop early appear to have ahemangioblast fate, whereas those that develop later generate thecardiac lineage and possibly other populations. The GFP⁺/Flk-1⁺populations appear to have lost the cardiomyocyte potential and may berestricted to the hemangioblast lineages. Given these findings, theearly developing (day 2.75-3.0) GFP⁺/Flk-1⁻ cells are referred to aspre-hemangioblast mesoderm whereas the population that develops betweenday 3.5 and 4.0 are referred to as pre-cardiac mesoderm. The day 3.0-3.5GFP⁺/Flk-1⁺ populations generate BL-CFC, whereas those isolated fromlater stage EBs (day 4.0) contain primitive erythroid progenitors,indicating the onset of hematopoietic commitment. Given thisdevelopmental potential, the GFP⁺/Flk-1⁺ population is referred to ashemangioblast mesoderm.

Examples 5 and 6 demonstrate that GFP+ cells isolated from differentaged EBs have different developmental potential. As indicated in theprevious examples, GFP⁺/Flk-1⁻ cells from day 3 EBs efficiently generateboth hematopoietic and endothelial lineages. These cells did not giverise to cardiotiyocytes (heart tissue) as demonstrated by the lack ofbeating cell masses. In contrast, GFP⁺ cells from day 4 EBs gave rise tofew Flk-1⁺ cells and BL-CFC following culture. This population did,however, generate cells of the cardiomyocte lineage. These findingsdemonstrate that the GFP⁺ (brachyury⁺) fraction isolated from differentaged EBs have become patterned to distinct populations with differentdevelopmental fates. In addition to the conditions used and potentialsobserved in the foregoing examples, other potentials may be observed byaltering conditions and additives.

Example 8 Role of Serum-Derived Factors

To assess the role of serum in the development of brachyury⁺ cells, EBswere differentiated the absence of serum. EBs did develop under theseconditions, although they were somewhat smaller than those found innormal conditions. In the absence of serum, no GFP⁺ cells were detectedwithin these EBs, indicating that mesoderm was not induced under theseconditions (FIG. 8). Significant numbers of GFP⁺/Flk-1⁻ and GFP⁺/Flk-1⁺cells did develop when serum was added to the cultures. These findingsclearly demonstrate that components found within serum are able toinduce the development and differentiation of brachyury⁺ cells. As afirst step in identifying factors that might play a role in thisprocess, BMP4 (20 ng/ml) was added to the developing EBs in the serumfree cultures. At this concentration, BMP4 did induce a significantpopulation of brachyury⁺ cells within 3 days of differentiation.However, in contrast to the serum, BMP4 did not support the developmentof the GFP⁺/Flk-1⁺ population in this period of time. To determine ifBMP4 could induce GFP⁺/Flk-1⁺ from GFP⁺/Flk-1⁻ cells that were inducedin the presence of serum, GFP⁺/Flk-1⁻ cells were isolated from EBsdifferentiated for three days in the presence of serum. These cells werereaggregated in medium alone, in medium with serum or in medium withBMP4. As shown in the lower row of FIG. 8, GFP⁺/Flk-1⁻ cells did notdifferentiate substantially when reaggregated in the absence of serum.As expected, the same population generated a large GFP⁺/Flk-1⁺population when serum was added to the cultures. Consistent with thefindings in the primary differentiation cultures, BMP4 was unable toinduce the development of significant numbers of GFP⁺/Flk-1⁺ cells fromthe cultured GFP⁺/Flk-1⁻ cells.

FIG. 9 summarizes the stages in mesoderm development based upon theforegoing examples. Step #1 represents mesoderm induction and patterningto a pre-hematopoietic and endothelial (pre-hemangioblast) fate. Step #2represents specification to the hematopoietic and endothelial lineages.Steps #3 and #4 represent patterning to the pre-cardiac fate.

Example 9 Isolation and Characterization of Endoderm Potential of CellPopulations

Studies using model systems such as Xenopus and Zebrafish have suggestedthat mesoderm and endoderm develop from a common precursor populationknown as mesendoderm. To determine whether or not a mesendoderm stage ofdevelopment does exist in EBs, conditions for the development of theendoderm lineage were established. As a first step in establishingculture conditions for the development of this lineage, the amount ofserum in the differentiation cultures was varied. EBs were generated inthe presence and absence of serum (SP34 and then IMDM plus SR) andassayed at different stages for expression of genes associated withectoderm, endoderm and mesoderm development. For the ectoderm lineage,development of the neuronal lineage was assessed analyzing expression ofPAX6, Wntl, neruoD and neurofilament (NFL). These genes are known to beexpressed at different stages of neruonal development. The early stagesof endoderm development were monitored by expression of HNF3β. Toevaluate later stages of endoderm development and specification, genesinvolved in liver development were analyzed. These included Hex,α-fetoprotein (AFP), HNF4, Albumin (Alb), α-1-antittrypsin (AAT) andtyrosine aminotransferase (TAT). Mesoderm development was monitored byexpression of brachyury and GATA-1. In addition to gene expressionpatterns, neuronal development was assayed by monitoring neuriteoutgrowth from EBs. The neuronal nature of these neurites wasdemonstrated by βIII-Tubulin expression. Mesoderm development was alsoassessed by enumeration of hematopoietic progenitors in the EBs. FIG. 10shows the impact of serum on the developmental potential of EBs over a10-day differentiation period. In the presence of serum (serum) there islittle neuro-ecotderm differentiation as demonstrated by the lack ofexpression of the genes associated with development of this lineage.HNF3β is expressed at early stages of differentiation (day 2-3) and thendownregulated. As expected, GATA-1 is expressed in the EBs generatedunder these conditions. The pattern of expression of these genes wasbasically reversed in EBs grown in the absence of serum (serum-). TheseEBs expressed all the genes associated with neuroectoderm, but did notexpress the mesoderm/hematopoietic marker GATA-1. HNF3β was expressed inlate stage EBs (day 10) grown under these conditions. The patterns ofbrachyury expression as monitored by GFP expression were consistent withthese findings. EBs generated in the presence of serum generated asubstantial brachyury⁺ population that was present between days 2 and 5of differentiation (FIG. 11, −B-line). Brachyury was not detected in EBsgrown in the absence of serum (FIG. 11, −H-line). Hematopoieticprecursor assays confirmed these findings. EBs generated in serumcontained precursors (FIG. 12, speckled bar), whereas those grownwithout serum did not (FIG. 12, solid bar, not visible). Finally,evaluation of neurite potential of the EBs was consistent with thesevarious analyses. None of the EBs grown in serum generated neurites. Incontrast, 85% of those generated in the absence of serum displayed thisactivity (FIG. 13). Taken together, these findings demonstrate theimportance of culture conditions (serum) for the generation of specificlineages. They also demonstrate that neither serum complete- norserum-free-conditions were optimal for endoderm development.

The strong upregulation of HNF3β in the early stage EBs generated inserum suggested that serum might be important for the establishment, butnot the maturation of the endoderm lineage. To test this possibility,EBs were initiated for 2 days in the presence of serum and then switchedto serum-free conditions (SR). As shown in FIG. 14, EBs generated underthese conditions (serum+/−) expressed HNF3β between days 3 and 5 ofdifferentiation. AFP was upregulated at day 5 of differentiation. GATA-1expression levels were reduced compared to those found inserum-stimulated EBs. Next, day 6 EBs generated under the serum+/−conditions were plated in tissue culture grade dishes (to allow them toadhere) in the presence of the growth factor bFGF. The dishes werecoated with either gelatin or matrigel to determine if substrate had anyimpact on further endoderm differentiation. Five days later, the mediumwas changed and additional factors were added to these cultures topromote the development of the liver lineage. The experimental outlineand data are shown in FIG. 15. In this experiment AFP was not expressedat the day 6 EB stage. Its levels of expression were upregulated whencultured in the presence of bFGF on either gelatin of matrigel. Lowlevels of ALB were also detected at this stage. ALB expression increasedfollowing the additional culture period in all conditions tested. AATand TAT were also expressed following the last culture step. The highestlevels of TAT were found when EB-derived cells were cultured in thepresence of bFGF and Dex. These findings clearly indicate that it ispossible to generate cells of the endoderm lineage and that underappropriate conditions, they will give rise to cells that express genesassociated with the developing liver.

It was further determined if these endodermal cells developed frombrachyury⁺ or brachyury⁻ cells. To address this question, GFP (Bry)⁺ andGFP (Bry)⁻ cells were isolated from day 2.5 EBs by cell sorting. Thesepopulations were allowed to reaggregate and cultured as clusters untilday 6. On day 6, they were moved to the tissue culture grade dishes inmedium with bFGF for 4 days (total of 10 days). Gene expression analysisindicated that cells, which express HNF3β, segregated to the GFP⁺fraction (d2.5) (FIG. 16). With time in culture, this gene was expressedin cells generated from the GFP⁻ fraction. This likely reflects the factthat at later stages of expression HNF3β is expressed in non-endodermalpopulation. AFP, HEX, ALB and HNF4 were all expressed in derivatives ofthe GFP⁺ fraction, but not in cell populations generated from the GFP⁻cells. In contrast, PAX6 and neuroD, markers of neuroectoderm, werefound predominantly in cells generated from the GFP⁻ fraction. Thesefindings indicate that the endoderm lineage is established from abrachyury⁺ population, which also gives rise to the mesodermal lineage,and that these lineages derive from a common precursor, the mesendoderm.

To further evaluate the liver potential of the brachyury expressingcells, cell populations derived from both the bry⁺ and bry⁻ fractionswere analyzed for expression of genes representing early hepatocytedevelopment such as α-fetoprotein (AFP), albumin (ALB) and transthyretin(TTR) and genes indicative of maturation of the lineage includingalpha1-antitrypsin (AAT), tyrosine aminotransferase (TAT) and carbamoylphosphate synthetase I (CPase). β-actin expression was used as acontrol. Cells were analyzed prior to sorting and subsequent to sortinginto bry⁺ and bry⁻ populations. Fetal liver and adult liver controlswere also analyzed. Expression of all of these genes was restricted tothe cells derived from the bry⁺ population, indicating that cells withhepatocyte characteristics develop from brachyury expressing cells.

Example 10 Kinetics of Mesoderm and Endoderm Development in EBs

The preliminary kinetic analysis described in Example 6 demonstratedthat subpopulations of mesoderm with distinct developmental fates weregenerated in a defined temporal fashion. A more detailed kineticanalysis showed the dynamic development of the GFP⁺Flk1⁻ (hereafterreferred to as the GFP⁺ population) population between days 2.5 and 4.0of differentiation (FIG. 18). When isolated and reaggregated, the day2.5 and 3.0 GFP⁺ fractions generated blast cell colonies (indicated ashemangioblast potential in FIG. 19) that represent the earliest stagesof hematopoietic and endothelial commitment (FIG. 19). This populationof GFP⁺ cells displayed little cardiomyocyte (cardiac) potential. Incontrast to the early GFP⁺ cells, those isolated from day 3.5 EBs showedsignificantly reduced BL-CFC potential, but were efficient atdifferentiating into cardiomyocytes. GFP⁺ cells isolated from day 4 EBsdid not give rise to cardiomyoctes and had little capacity to generateBL-CFC, suggesting they may be fated to some other mesodermal lineage.Gene expression analysis supported these functional assays anddemonstrated molecular differences between the four GFP⁺ fractions (FIG.20). While some genes were expressed in all populations, others showedintriguing differential patterns. Wnt3a, a gene thought to be importantfor hematopoietic development and inhibitory for cardiacdifferentiation, was expressed in the day 2.5 and 3.0 populations anddown regulated in the day 3.5 and 4.0 cells. This pattern is consistentwith the change in developmental potential of these populations fromhematopoietic/vascular to cardiac muscle. A second pattern of interestis that of the gene Mix1-1. This gene, which plays a role in thedevelopment of mesoderm and endoderm, was expressed in the day 2.5 and3.0 GFP populations but not in the day 3.5 and 4.0 fractions. Takentogether, the findings of this example clearly demonstrate that mesodermpopulations with different developmental potential are generated in adefined temporal pattern within the EBs. In addition, expressionanalysis of the mesoderm/endoderm gene Mix1-1 indicates that cells withendoderm potential are also generated at a specific time, namely betweenday 2.5 and 3.0 of differentiation.

To further investigate the kinetics of endoderm development, GFP⁺ cellsfrom day 3.0 and 4.0 EBs were isolated and cultured under conditionsthat promote the differentiation into hepatocyte-like cells. As shown inFIG. 21, GFP⁺ cells (+/−) isolated from day 3.0 but not those from day4.0 displayed endoderm potential as defined by expression of HNF3β.These findings indicate that endoderm is generated within the GFP⁺population at a specific period of time, prior to day 4 ofdifferentiation.

Example 11 Developmental Potential of GFP⁺ Populations In Vivo

To further evaluate the endoderm potential of the GFP⁺ population, GFP⁺and GFP⁻ day 2.5 EB cells were cultured for 14 day days under conditionsknown to promote hepatocyte differentiation and then transplanted underthe kidney capsule of recipient SCID-beige mice. Several mice weresacrificed immediately following the transplant, the kidney with thegraft was sectioned and the sections were stained with antibodiesagainst Hep1 and AFP. Hep1 is a specific marker of hepatocytes whereasAFP is expressed in definitive endoderm and immature cells of thehepatocyte lineage. Some of the cells within the section stainedpositive for Hep1, whereas other cells, in the vicinity of the Hep1⁺cells were found to express AFP. No Hep1⁺ or AFP⁺ cells were found inthe graft of the GFP⁻ negative cells. These findings support PCR dataand demonstrate that there culture conditions support the development ofcells with characteristics of immature hepatocytes, as defined byexpress of Hep1.

While these transplantation experiments demonstrate the presence ofhepatocyte-like cells in the grafts immediately followingtransplantation, it was difficult to monitor the maturation of thesepopulations over time, as the transplanted tissues generated tumor-likemasses known as teratomas. The teratomas likely develop fromcontaminating undifferentiated ES cells or from GFP⁺ primordial germcells that are known to be of mesoderm origin and to express brachyury.

Example 12 Induction of Mesoderm and Endoderm by Activin

To further enrich for cells with endodermal potential, the effects ofgrowth factors known to induce this cell population in other modelsystems were tested. Studies in Xenopus have shown that activin willinduce both mesoderm and endoderm from ectoderm in culture. Ofparticular interest was the observation that activin behaved as amorphogen in this model in that it induced different cell types atdifferent concentrations used. To determine if activin displayed similarpotential in the ES/EB system, it was added to the EB cultures using thefollowing protocol. ES cells were differentiated for 2 days in Stem Pro34 medium without serum. At this stage, the developing EBs wereharvested and recultured in IMDM supplemented with serum replacement(serum free) and activin at a concentration of 100 ng/ml. EBs wereharvested at different days and assayed for GFP⁺ expression andexpression of genes indicative of ectoderm, mesoderm and endodermdevelopment. As shown in FIG. 22A,B this amount of activin-inducedbrachyury as measured by GFP. While the kinetics of GFP induction wasdelayed compared to EBs differentiated in serum, this concentration ofactivin did induce substantial numbers of brachyury positive cells (60%)by day 6 of differentiation. Molecular analysis indicated that theactivin-induced cells expressed a broad spectrum of genes associatedwith endoderm development including HNF3β, Mix1-1, Sox17, Hex-1, andpdx-1 (FIG. 23C). Induction of pdx-1 is of interest, as this gene isessential for pancreas development. Genes associated with hematopoieticdevelopment, such as GATA-1 and those indicative of neuroectodermdifferentiation such as PAX6 were not induced by activin.

To determine if activin displayed morphogenic properties in the ESdifferentiation model, different concentrations of the factor were addedto the EB cultures. As little as 1 ng/ml of activin induced GFP⁺expression (10% of the total population) by 7 days of culture (FIG.23A). The frequency of GFP⁺ cells increased to 40% in culturesstimulated with 3 ng/ml and reached plateau levels of greater than 50%at 30 ng/ml. Gene expression analysis of these populations indicatedthat different concentrations of activin did induce differentdevelopmental programs. EB differentiated in the presence of 1 or 3ng/ml of activin showed weak, if any, expression of the genes indicativeof endoderm development (FIG. 23A). HNF3β, Sox17, Hex-1 were all inducedin cultures stimulated with 10, 30, or 100 ng/ml of activin. Pdx-1expression required the highest amount of activin and was induced bestin EBs stimulated with 100 ng/ml. Neither GATA1 nor c-fms was expressedat any concentration of activin. The pattern of PAX6 expression was thereverse to that of the endoderm genes and was downregulated withincreasing concentrations of activin.

Activin-induced EBs did not express genes associated with hematopoieticcommitment indicating that this developmental program was not induced.To further evaluate the hematopoietic potential of these EBs, they wereanalyzed for progenitor potential. As expected from the molecularanalysis, these EBs contained no appreciable numbers of primitive (Ep)or definitive (mac/mix) hematopoietic progenitors (FIG. 24A). When theseactivin induced EBs were further stimulated with serum for 2.5 days,however, they did generate some hematopoietic progenitors, indicatingthat they do contain mesodermal potential (FIG. 24B).

PCR analysis demonstrated that activin induced expression HNF3β as wellas other genes known to be involved in endoderm differentiation. Tobetter estimate the proportion of endodermal progenitors in theactivin-induced EBs, cells from cultures stimulated with 100 ng/mlactivin were stained with antibodies against HNF3β and Hex1. EBs fromun-induced cultures were used as controls (Activin-). A significantportion of the activin induced population (estimated at 50-60% of total)expressed both HNF3β and Hex1. None of the cells in the un-induced EBsexpressed these proteins. These findings clearly demonstrate that asubstantial number of cells within these EBs are of the endodermlineage.

To further investigate the potential of these activin-inducedpopulations, GFP⁺ cells were isolated from EBs stimulated with 3 ng or100 ng and cultured further (14 day total) in hepatocyte conductions. Asshown in FIG. 25, only cells from the 100 ng cultures differentiatedinto cells that expressed albumin consistent with liver differentiation.Taken together, the findings from these studies indicate that activinfunctions as a morphogen in the ES/EB system and that high concentrationare required for endoderm induction.

If the teratomas generated by the serum-induced brachyury⁺ cellsresulted from the presence of primordial germ cells, it is possible thatthe activin induced cells may represent a better source of progenitorsfor transplantation, as the germ-cell program may not be induced underthese conditions. To test this hypothesis, GFP⁺ and GFP⁻ cells wereisolated from EBs induced with 100 ng/ml of activin and cultured for 14days to promote the differentiation of hepatocyte-like cells. Followingthis culture period, the cells were harvested and transplanted to thekidney capsule of recipient animals. Three weeks followingtransplantation, the mice were sacrificed and the kidneys analyzed.Results are shown in FIGS. 26A-C. All mice engrafted with GFP⁻ cellsdeveloped large, multilineage teratomas, consisting of cells from allthree germ layers. In contrast, no teratomas were detected in theanimals transplanted with GFP⁺ cells. These cells give rise todifferentiated cell masses consisting of endodermal and mesodermalderived tissues including gut epithelium, bone and skeletal muscle. Insome instances, skin was also observed in the graft from the GFP⁺ cells,suggesting that this lineage might also develop from a bry⁺ cell. Thesefindings indicate that it is possible to generate GFP⁺ populations thatgive rise to differentiated tissue without forming teratomas followingtransplantation.

Example 13 Developmental Potential of Bry⁺/c-Kit⁻ and Bry⁺/c-Kit⁺ Cells

The foregoing examples clearly indicate that the hepatocyte lineagedevelops from a bry⁺ cell population that has both mesoderm and endodermpotential. Analysis of the bry⁺ fraction revealed that a subpopulationof these cells expressed the receptor tyrosine kinase c-kit (FIG. 27A)and that this population was distinct from those cells that expressedFlk-1. To determine if c-kit expression could be a useful marker for thesegregation of cell with endodermal potential, bry⁺/c-kit⁻ (+/−) andbry⁺/c-kit⁺ (+/+) cells from day 3 serum-stimulated EBs were assayed forhepatocyte potential. As shown in FIG. 27B, HNF3β, AFP and ALBexpressing cells were all derived from bry⁺/c-kit⁺ population. Toestimate the endodermal potential of this fraction, sorted cells wereplated onto glass coverslips and stained with an antibody to HNF3β.Greater than 80% of the bry⁺/c-kit⁺ cells expressed HNF3β protein,whereas less than 10% bry⁺/c-kit⁻ were positive. These findings indicatethat endodermal progenitors express both brachyury and c-kit and thatthis population is highly enriched for cells with endoderm potential.Isolation of cells based on brachyury and c-kit expression provides anovel strategy for the isolation of endodermal progenitors.

Example 14 Developmental Potential of Activin-Induced Cells

The PCR analysis presented in FIG. 23 indicates that differentconcentrations of activin induce different developmental programs andthat cells with endodermal potential are induced with the highest levelsof this factor. To quantify the differential response of activin, cellsstimulated with different concentrations of activin were adhered tocoverslips and stained with the anti-HNF3β antibody. More than 50% ofthe entire EB population stimulated with 100 ng/ml of activin expressedHNF3β whereas only 10% of the cells stimulated with 3 ng/ml werepositive. Only background levels of staining were observed in thenon-stimulated population. These findings demonstrate that highconcentrations of activin can stimulate a robust endodermal program,representing a significant portion of entire EB population.

As a further assessment of the potential of activin treated cells, day 6EBs differentiated in the presence of different concentrations of thisfactor were transferred to serum replacement media for 4 days and thenreplated in hepatocyte conditions for an additional 4 days. At day 14 ofculture, the cells from each group were harvested and subjected to PCRexpression analysis. Expression of Myf5 and skeletal actin weremonitored to evaluate skeletal muscle development representing anadditional mesoderm-derived lineage. Surfactant protein C (SP-C), alung-specific gene, was included as a marker of endoderm differentiationin addition to AFP and ALB. As shown in FIG. 28, Myf5 and skeletal actinwere expressed in cultures stimulated with as little as 1 ng/ml ofactivin and this expression was detected over a broad range of factorconcentrations. Expression of both genes was, however, downregulated atthe highest concentration of activin (100 ng/ml). Cultures stimulatedwith low amounts of activin contained groups of cells with themorphology of skeletal muscle. Immunostaining demonstrated that thesecells expressed both skeletal myosin and α-actinin, indicating that theyare of the skeletal muscle lineage. Evaluation of the proportion ofreplated EBs that generated skeletal muscle outgrowths was consistentwith the gene expression analysis, as those stimulated with 3 and 10ng/ml displayed the most robust skeletal muscle development as depictedin FIG. 28. The expression patterns of the three endodermal genesdiffered from that observed for the skeletal muscle genes. None wereexpressed at low activin concentrations and all were readily detected incultures stimulated with the highest concentrations of the factor.Expression of PAX6 was restricted to untreated cultures and thosestimulated with low concentrations of factor. The findings from thisanalysis confirm and extend those from Example 12 hereinabove indemonstrating that different concentrations of activin induce differentdevelopmental programs, with low concentration favoring a mesodermalfate and high concentrations an endoderm fate. In addition, theseresults indicate that the endodermal cells induced by activin are ableto differentiate and give rise to cells with hepatocyte and lungcharacteristics.

Brachyury positive and negative populations isolated from EBs stimulatedwith low and high concentrations of factor were also analyzed forexpression of the skeletal muscle and endoderm genes. As shown in FIG.29, both myf5 and skeletal actin expression were restricted to thepopulation generated from the bry⁺ population isolated from EBsstimulated with 3 ng/ml of activin. Similarly, the endoderm genes wereexpressed in the brachyury⁺-derived cells isolated from EBs generated inthe presence of 100 ng/ml of factor. These findings further demonstratethat both mesoderm and endoderm develop from brachyury⁺ cells.

Example 15 Generation of HNF-CD4 Targeted ES Cells

In order to track and isolate endodermal as well as mesodermalpopulations during EB differentiation, ES cells which already have GFPtargeted to the brachyury locus were targeted with human CD4 (hCD4)protein into the HNF 3 β locus. HNF3β is known to be expressed in mostendodermal cell types, starting very early during embryonic development.Kaestner et al. (1994) Genomics 20:177-85. The targeting constructcontains the human CD4 cDNA, truncated at amino acid number 424, andlacks most of the intracellular domain. This cell surface molecule waschosen because it cannot transduce signals and there are readilyavailable antibodies for flow cytometry, which recognize hCD4 and willnot cross react with endogenous mouse CD4. The vector also contains tworegions of homology to the mouse HNF 3 β locus, the bovine growthhormone poly(A) sequence, and a loxp flanked puromycin cassette asdepicted in FIG. 30A. The diptheria toxin A (DTA) gene was included inthe 5′ end of the targeting vector to select against random integration.The targeting vector was constructed as follows.

The short arm and long arm of homology were obtained from a publishedHNF3B targeting vector. Weinstein et al. (1994) Cell 78: 575-588. Thesame long arm was used while a 1.8 kb fragment of the short arm was usedto create the hCD4 knock in vector. The short arm contains part of theHNF3B promoter, exon 1, intron 1, and part of exon 2, ending just beforethe endogenous ATG initiation site. The long arm consists of a 7 kbsequence starting 3 kb downstream of the end of the last exon. Usingstandard molecular biology techniques, hCD4 with a kozak sequencefollowed by the bovine growth hormone poly(A) sequence along with apuromycin cassette was cloned in between the two arms of homology asshown in FIG. 30A.

The GFP-Bry ES cells described in example 2 were electroporated with theHNF3 β targeting vector. The cells were then selected with puromycin andclones were picked and expanded. Genomic DNA was isolated, cut with therestriction enzyme Sac I, and a Southern blot analysis was performedwith a probe upstream of the endogenous ATG as indicated in FIG. 30B.The wild type allele gives a 3.3 kb band while the targeted allele givesa 2.8 kb sized band. Of 48 clones screened, 2 targeted clones weregenerated, corresponding to a 4.2% targeting efficiency. Both targetedclones were transiently transfected with a modified Cre recombinaseexpression vector to remove the puro gene. The targeted clones arereferred to hereinafter as CD4-HNF/GFP-T ES cells.

HNF3B is expressed transiently in EBs during serum induceddifferentiation. To determine whether hCD4 expression in CD4-HNF/GFP-TES cells accurately reflects expression of the HNF3β gene during EBdevelopment, CD4 versus GFP expression was assessed.

A typical expression pattern of GFP versus hCD4 during a 5 day seruminduced EB differentiation is shown in FIG. 3 IA. ES cells are low tonegative for hCD4 expression. Following differentiation, hCD4 expressionis sharply upregulated specifically in the GFP⁺ cells at day 3.Expression falls rapidly by day 3.5 and is almost undetectable by day 5.In contrast, GFP expression persists to day 4 and decreases by day 5.The mean fluorescence intensity for GFP and hCD4 expression is shown inFIG. 3 IB. It is seen that hCD4 peaks at day 3 and is lost quickly whileGFP peaks at days 3.5 to 4. Semi-quantitative RT PCR for HNF3B andBrachyury expression was also performed on the samples (FIG. 31C). ThePCR for the endogenous genes follows what is seen using flow cytometryfor GFP and hCD4. HNF3B and Brachyury are both expressed by day 3, whileHNF3B expression decreases by day 3.5 and is undetectable by day 5.Brachyury expression continues at high levels up to day 4. These dataconfirm that hCD4 is mirroring the expression of endogenous HNF3B. Inaddition, hCD4 expression is seen to decline along with HNF3B RNAlevels, suggesting that the half live of the hCD4 protein is notexcessively long and can be used to accurately select cells expressingHNF3B. It is also seen that all GFP⁺ cells are initially hCD4⁺, but thenrapidly lose expression of hCD4. This data is suggestive of amesendoderm precursor to both endoderm and mesoderm, supporting the datain example 9. This population has been demonstrated in lower organismssuch as c. elegans but has not been established in mammals. Theconditions used above have been selected for robust mesoderm induction.It is possible that mesendoderm is formed but is rapidly converted intomesoderm.

Example 16 EB Differentiation as a Model for Primitive Streak Formation

During embryogenesis, both the mesoderm and endoderm lineages arederived from a structure called the primitive streak. Beddington andRobertson (1999) Cell 96: 195-209. The primitive streak is characterizedby expression of specific genes such as Brachyury, which is expressedalong the whole streak. HNF3B is also expressed in the streak, though ithas only been detected in the anterior section. HNF3B expression asdemonstrated by hCD4 was seen to be heterogeneous in the Bry-GFP⁺population. It is possible that serum induction allows development of agroup of primitive streak like cells, with the GFP⁺ HNF3B^(lo) cellsrepresenting the posterior streak while the GFP⁺HNF3B^(hi) cellscorrespond to the anterior streak.

To determine if GFP and hCD4 expression could be used to separateposterior and anterior primitive streak like cells from CD4-HNF/GFP-T EScells, EBs differentiated for 3.25 days were sorted into threepopulations: GFP⁺ hCD4^(lo), GFP⁺ hCD4^(med), and GFP⁺hCD4^(hi) (FIG.32A). There are many genes that are recognized to be expressedasymmetrically in the primitive streak. RT PCR was performed on thesorted populations for genes known to be expressed in either theposterior or anterior primitive streak (FIG. 32B). The posteriorprimitive streak specific genes Fgf8, EvxI, HoxBI, and Tbx6 were allexpressed at higher levels in hCD4^(lo) and hCD4^(med) populationscompared to hCD4^(hi) cells. In contrast, the anterior primitive streakgenes Chordin, Cerl, and Gsc are all expressed at high levels inhCD4^(hi) cells, low to negative in hCD4^(med) cells, and not at all inhCD4^(lo) cells. HNF3B expression was also examined to verify that thehCD4 sorting isolated cells expressing different levels of the HNF3Bgene. The highest levels of HNF3B were seen in the hCD4^(hi) populationwith decreasing amounts in the hCD4^(med) and hCD4^(lo) populations asexpected. To confirm that the expression patterns in the various hCD4populations is an accurate representation of gene expression in theprimitive streak, the primitive streaks of day 7.25 mouse embryos weredissected and analyzed for gene expression. Mice embryos were used thatwere derived from the GFP-Bry ES cells. The anterior, middle, andposterior primitive streak tissues were then dissected under afluorescent dissection scope to ensure that the tissues obtained werefrom the GFP⁺ (primitive streak) portions of the embryo. FIG. 32C showsthe results of RT PCRs performed from the dissected streak tissues.These data indicate that the sorted hCD4 populations show similarrelative gene expression profiles for the anterior and posterior genesexamined. These data indicate that the expression of hCD4 and GFP inCD4-HNF/GFP-T ES cells can be used to isolate cells with gene expressionpatterns indicative of the anterior and posterior primitive streak.

Example 17 Function Potentials of Different Populations of hCD4Expressing Cells

Lineage tracing experiments have demonstrated that the anterior andposterior primitive streak develop into distinct lineages. The posteriorstreak gives rise to the yolk sac and hematopoietic cells while theanterior streak forms endoderm and anterior mesoderm such as cardiactissue. Robb and Tarn (2004) Sem. Cell & Dev. Biol. 15: 543-554. Todetermine if the primitive streak like cells from EBs behavefunctionally like the endogenous primitive streak, CD4-HNF/GFP-T EScells were differentiated for 3.25 days, sorted for GFP⁺ hCD4^(lo) andGFP⁺ hCD4^(hi) populations and assayed for hematopoietic, cardiac, andendoderm potential. For all assays, the sorted populations were allowedto reaggregate for 48 hours in serum free conditions. FIG. 33 A showscardiac potential of sorted cells which were plated onto matrigel afterreaggregation. Individual EBs were scored for the presence of beatingcells 24-48 hours after adhering. Beating EBs were only seen from thehCD4^(hi) population at this time point. FIG. 33B demonstrateshematopoietic potential of the sorted populations. Cells weretrypsinized after reaggregation and plated in methylcellulose withhematopoietic growth factors. The presences of hematopoietic colonieswere counted 5-7 days later. The majority of all hematopoietic colonieswere found in the hCD4^(lo) population. FIG. 33C shows RT PCR forendoderm specific genes of the sorted populations which were plated ontomatrigel in serum free conditions after reaggregation. Only thehCD4^(hi) cells expressed the endodermal genes HNF4, Pdxl, AAT, ALB, andHNF3B after 5 days on matrigel. These data demonstrate that theexpression of hCD4 and GFP can be used to segregate populations withdifferent developmental potentials. These potentials are consistent withhCD4^(lo) and hCD4^(hi), cells being similar to the posterior andanterior primitive streak respectively.

Example 18 Activin Signaling and Wnt Signaling are Both Required for ESDerived Primitive Streak Like Cells

Studies in mice have demonstrated the importance of Wnt signaling aswell as nodal/activin signaling in gastrulation and endoderm/mesoderminduction. Liu et. al. (1999) Nat. Genet. 22: 361-4. Example 12hereinabove demonstrates that activin can induce both mesodermal andendodermal populations from ES cells in serum free cultures. Todetermine the effects of activin and Wnt signaling on thedifferentiation of ES derived primitive streak like cells, CD4-HNF/GFP-Tcells were differentiated in serum free culture with either recombinantWnt3a or activin A. Both activin and Wnt can initially induce GFP⁺ CD4⁺cells, though Wnt displays a faster kinetics (FIG. 34A). Activinstimulation leads to high levels of CD4 expression that persist afterloss of GFP-Bry expression at day 6. In contrast, GFP⁺ cells inducedwith Wnt have lower levels of CD4 and rapidly lose expression by day 5.activin also induces robust expression of other anterior primitivestreak markers such as Gsc and Cerl while Wnt does not (FIG. 34B). Wntdoes however stimulate expression of the posterior primitive streakmarkers HoxBI and Tbx6, while activin only induces Tbx6. These datasuggest that activin induces ES anterior primitive streak cells whileWnt induces ES posterior primitive streak cells. At day 6, EBs that werestimulated with either activin or Wnt3a were plated on matrigel in serumfree media to allow further development. After a further 6 days, thesecultures were harvested and endodermal gene expression was determined byRT-PCR (FIG. 34C). activin induction allowed development of cellsexpressing the endodermal genes Alb, Aat, HNF4, and Pdxl, confirmingfindings in example 12. However, the Wnt3a induced cultures did notexpress any of these markers suggesting that activin signaling isrequired for endoderm development.

Considering that Wnt stimulation induces nodal and activin treatmentinduces Wnt3 (FIG. 34B), it is unknown whether activin/nodal signalingor Wnt Signaling alone can induce primitive streak formation or whetherboth factors are required together. CD4-HNF/GFP-T ES cells were inducedwith either activin or Wnt in conjunction with specific inhibitors ofthese pathways. DKK1 was used to inhibit Wnt signaling while the smallmolecule inhibitor SB-431542 (SB) was used to block Nodal/TGFβsignaling. DKK1 functions by binding the Wnt co-receptors, LRP5 andLRP6, which are essential for activation of the βcatenin signalingpathway. SB is a specific inhibitor of the receptors ALK4, 5, and 7 andthus is able to block TGFβ, activin, and Nodal signaling. It does not,however, block BMP signaling. Inman et. al. (2002) MoI. Pharmacol. 62:65-74. Both inhibitors completely blocked the development OfGFP⁺ CD4⁺cells with either inducer (FIG. 34A). The primitive streak markers Gsc,Cerl, HoxBl, and Tbx6 were also absent or greatly reduced (FIG. 34B).This indicates that Wnt and activin/nodal signaling are requiredtogether to induce ES derived primitive streak like cells. Therefore, toinduce mesoderm and endoderm germ layers and their derivative tissues,both activin/Nodal and Wnt signals are required initially.

Example 19 Activin Signaling and Wnt Inhibition can Induce EndodermDifferentiation

Example 12 demonstrated that high levels of activin could induceendodermal gene expression from EBs differentiated in completely serumfree conditions. Nodal, a key molecule involved in mesoderm/endoderminduction in vivo, signals through the same receptors as activin. It isalso known that Nodal as well as inhibitors for Wnt signaling areexpressed preferentially in or around the anterior primitive streak.Considering that the endoderm forms from the anterior primitive streak,it is possible that nodal/activin signaling in combination withinhibition of Wnt signals induces endoderm formation. To test thishypothesis, CD4-HNF/GFP-T ES cells were differentiated for 3.25 days inserum and GFP⁺ hCD4^(med) cells were sorted and reaggregated in serumfree media with no factor, DKK1 (Wnt inhibitor), activin, or activin andDKK1. The effect of these culture conditions on hCD4-HNF3β and GFP-Brylevels were analyzed by flow cytometry (FIG. 35A). With serum free mediaalone, hCD4 expression was lost after 1 day reaggregation. Inhibition ofWnt signaling by the addition of DKK1 allowed some cells to retain lowlevels of hCD4. Addition of activin allowed the retention of HNF3βlevels similar to newly sorted cells. Lastly, activin and DKK1 workedsynergistically to increase hCD4 levels almost one log over the levelsafter sorting. GFP levels were lost for all of the conditions tested,suggesting that differentiation was proceeding in all cultures but thatthe cultures expressing more hCD4 were differentiating into endodermallineages. To further test this idea, sorted cells reaggregated for twodays in the culture conditions above were washed with serum free mediawithout factors and plated onto martrigel in serum free media. After 5days, the cultures were harvested and gene expression determined by RTPCR. FIG. 35B shows the expression of several endoderm genes specificfor the pancreas, liver, and intestine. None of these genes weredetected in either media alone or DKK1 treated cultures. Very low levelsof just two of the genes, Pdxl and ALB were detected in the culturestreated with activin alone. Treatment with both activin and DKK1 led tohigh expression of Pdxl, AAT, HNF4, ALB, Cdx2, and IFABP. These datademonstrate that the combination of signaling through the activinreceptor and inhibition of Wnt signals by DKK1 treatment inducesendoderm formation from a cell population that would not form endodermwhen cultured in media alone. These culture conditions have thepotential to allow the large scale production of endodermal derivedtissues such as the pancreas and liver. These experiments alsodemonstrate the utility of the CD4-HNF/GFP-T ES cells as a tool toassess endoderm development in a quantitative manner.

Example 20 Generation of Hepatocytes from Endoderm-Derived MouseEmbryonic Stem Cells

By using the GFP-Bry, hCD4-Foxa2 ES cell line described in Example 15hereinabove, endoderm cells were tracked by flow cytometry by their GFPand hCD4 expressions upon ES cell differentiation. (The terms HNF3β andFoxa2 are used interchangeably.

GFP-Bry, hCD4-Foxa2 ES cells were maintained in absence of serum andfeeder cells in a media consisting of Neurobasal, DMEM/F12, N2supplement, B27 with retinoic acid, 10% BSA, MTG (1.5×10″⁴M), Glutamin,LIF (1000U/ml) and BMP-4 (10 ng/ml). In these conditions, cells formtight colonies that remain undifferentiated. In order to induce theendoderm program, ES cells were harvested and cultured in presence ofactivin-A (50 ng/ml) in a serum-free media (IMDM, F12 media, B27 withretinoic acid, N2 supplement, BSA, MTG, glutamin, ascorbic acid) at lowdensity (35,000 cells/5 ml) in a ultra low attachment dishes to allowembryoid bodies (EB) formation. As the ES cells differentiate,expression patterns for GFP-Bry, hCD4-Foxa2 and the receptor for stemcell ligand c-Kit have been defined. A large cell population GFP-Bry⁺,hCD4-Foxa2^(high) and cKit^(high) (named +++) emerged at day 4, and wasassayed for its endoderm potential and hence hepatic fate versus theircounterpart population GFP-Bry+, HCD4^(low) and c-Kit⁻ (named +L-). Toaddress these issues, both populations were reaggregated for 2 days inpresence of the two cytokines known to specify endoderm cells into liverin the embryo, BMP-4 (30 ng/ml) and bFGF (10 ng/ml) in the serum freemedia indicated above. Zaret (2001) Curr Opin Genet Dev 11:568-574. Inaddition to the two cytokines, activin-A (50 ng/ml) has been added tomaintain an endoderm potential of the cells as opposed to a mesodermfate. To allow maturation of the hepatocyte progenitors (hepatoblasts),aggregates were then plated on gelatin-coated dishes in a media that isappropriate for hepatoblast/hepatocyte maturation and growth. This mediaconsists of the basic differentiation media indicated above supplementedwith VEGF (10 ng/ml), dexamethasone and other cytokines present in theBlock media known to promote hepatoblast/hepatocyte growth Those includehuman recombinant HGF (20 ng/ml), EGF (10 ng/ml), bFGF (10 ng/ml) andTGFalpha (20 ng/ml). Block et al., (1996) J Cell Biol 132:1133-1149. Sixdays later, +++ aggregates gave rise to numerous “hepatic colonies”attached to the dish as well as a large number of floating aggregatesthat did not attach. All +L-aggregates attached to the dish and formedvery few “hepatic colonies”, but no floating aggregates were seen in theculture. These results suggest that the day 4+++ population is highlyenriched in endoderm cells and demonstrate that the combination ofBMP-4, bFGF and activin-A promote strongly the specification of the day4+++ cells into hepatocytic lineage.

One day following plating on gelatin-coated dishes, ⅔ of the +++aggregates remained floating in culture, while the other ⅓ attached andformed hepatic colonies that were sometimes surrounded by other cells.At this early time point, many cells within the colonies expressedalbumin and AFP assayed by immunohistochemistry. The surrounded cellswere all CD31 positive cells suggesting that they are endothelial cells.

Later on at day 12, hepatic colonies grew larger. At this later timepoint, most of the cells within the colonies are AFP and albuminpositive, and most of the cells surrounding the colonies are thepresumptive endothelial CD31 positive cells. Immunohistochemistry forhCD4 showed that all the cells contained in hepatic colonies expressedfoxa2, and that this expression was restricted to the colonies. Theseresults confirmed the endoderm origin of the hepatic colonies.

Similarly, day 12 floating +++ aggregates grew much larger and wereprocessed for immunohistochemistry for AFP and albumin. Most of theaggregates were hollow, and cells lining up the aggregates expressed AFPand albumin, while the IgG control section was negative. In addition,AFP and albumin were also stained inside the aggregates, suggestingsecretion of both these proteins.

In order to further characterize the day 12+++ cultures, flow cytometryanalysis was performed on both attached cultures and floating aggregatesto quantify the endoderm-derived population (hCD4-Foxa2⁺), endothelialcell population (CD31+ or PECAM-I), hepatoblast/hepatocyte population(hCD4+/alb+, or hCD4+/AFP+) (FIG. 36A, 36B). 30% to 40% of the cellscontained in the attached cultures were positive for the endoderm markerhCD4-Foxa2, while 50% were CD31⁺ endothelial cells. Floating aggregatesconsisted of a larger (50% to 60%) of hCD4-Foxa2⁺ cells and a smallercell population of CD31+ endothelial cells (30%) (FIG. 36A). Most of thehCD4-Fpxa2+ cells were also albumin⁺ and AFP⁺ assayed byintra-cytoplasmic staining (FIG. 36B). Gene expression analysis byRT-PCR confirmed that the attached cultures (a) as well as the floatingaggregates (f) from day 12+++ cultures expressed faxa2, AFP and albumin(FIG. 36C).

Functional assays were performed to attest the hepatocyte identity. Acidperiod Schiff Assay indicates in the cytoplasm the presence of glycogenstorage that is characteristic of mature hepatocytes. Day 12+++ attachedcultures were stained directly in the dish and showed numerous cellswithin the colonies containing the typical red cytoplasmic staining forglycogen. Moreover, electro microscopy was performed on day 12+++floating aggregates. Most of the cells that lined up the floatingaggregates displayed hepatocyte characteristics including a largenucleus/cytoplasmic ration, large amount of glycogen storage andpresence of bile canaliculi.

Altogether these data indicate that hepatocytes are generatedefficiently from mouse ES cells by mimicking in vitro the developmentalprogression of the ES cells to an endoderm stage by activin-A induction,a liver specification step with the combination of BMP-4, activinA andbFGF, and finally a proliferation/maturation of thehepatoblast/hepatocyte population

1.-29. (canceled)
 30. A method of making a cell population enriched forendoderm cells comprising culturing pluripotent stem cells in thepresence of activin or nodal, and Wnt, for a time sufficient to providebrach⁺/HNF3β⁺ cells, and culturing said brach⁺/HNF3β⁺ cells in thepresence of activin and an inhibitor of Wnt signalling for a timesufficient to provide a cell population enriched for endoderm cells. 31.The method of claim 30 wherein the stem cells are cultured in theabsence of serum.
 32. The method of claim 30 wherein the brach⁺/HNF3β⁺cells are cultured in the absence of serum.
 33. The method of claim 30wherein the stem cells are human embryonic stem cells.
 34. The method ofclaim 30 wherein Wnt is Wnt 3d and the inhibitor of Wnt signaling isDKK-1.
 35. (canceled)
 36. The method of claim 30 wherein activin ispresent at a concentration of at least about 30 ng/ml.
 37. (canceled)38. (canceled)
 39. A cell population comprising at least about 10%hepatocytes produced by a method comprising the steps of (a) culturingpluripotent stem cells in the presence of activin to inducedifferentiation into endoderm lineages, (b) further culturing the cellsof step (a) in the presence of BMP-4 and bFGF and in the absence ofserum to induce differentiation into a hepatic lineage, and (c)culturing the cells from step (b) with one or more cytokines thatpromote hepatocyte growth to produce a population enriched forhepatocytes.
 40. (canceled)
 41. (canceled)
 42. The cell population ofclaim 39 wherein the cells are human cells.
 43. The cell population ofclaim 39 wherein activin is present at a concentration of about 30 ng/mlto about 100 ng/ml, BMP-4 is present at a concentration of about 10ng/ml to about 30 ng/ml, and bFGF is present at a concentration of about1.0 ng/ml to about 10 ng/ml. 44.-48. (canceled)
 49. The cell populationof claim 39 wherein the cytokines are selected from the group consistingof VEGF, human recombinant HGF, EGF, bFGF, and TGFalpha.
 50. The cellpopulation of claim 49 wherein VEGF is present at a concentration ofabout 10 ng/ml, human recombinant HGF is present at a concentration ofabout 20 ng/ml, EGF is present at a concentration of about 10 ng/ml,bFGF is present at a concentration of about 10 ng/ml, and TGFalpha ispresent at a concentration of about 20 ng/ml. 51.-54. (canceled)
 55. Amethod of screening for an agent that has an effect on hepatocytescomprising culturing hepatocytes in the presence and absence of an agentto be tested and determining whether the agent has an effect on thehepatocytes, wherein the hepatocytes are produced by a method comprisingthe steps of (a) culturing pluripotent stem cells in the presence ofactivin to induce differentiation into endoderm lineages, (b) furtherculturing the cells of step (a) in the presence of BMP-4 and bFGF and inthe absence of serum to induce differentiation into a hepatic lineage,and (c) culturing the cells from step (b) with one or more cytokinesthat promote hepatocyte growth to produce a population enriched forhepatocytes.
 56. The method of claim 55 wherein activin is present at aconcentration of about 30 ng/ml to about 100 ng/ml, BMP-4 is present ata concentration of about 10 ng/ml to about 30 ng/ml, and bFGF is presentat a concentration of about 1.0 ng/ml to about 10 ng/ml. 57.-61.(canceled)
 62. The method of claim 55 wherein the cytokines are selectedfrom the group consisting of VEGF, human recombinant HGF, EGF, bFGF, andTGFalpha.
 63. The method of claim 62 wherein VEGF is present at aconcentration of about 10 ng/ml, human recombinant HGF is present at aconcentration of about 20 ng/ml, EGF is present at a concentration ofabout 10 ng/ml, bFGF is present at a concentration of about 10 ng/ml,bFGF is present at a concentration of about 10 ng/ml, and TGFalpha ispresent at a concentration of about 20 ng/ml. 64.-67. (canceled)
 68. Amethod of hepatocyte replacement therapy comprising administering to asubject in need of such treatment a composition comprising hepatocytesproduced by a method comprising the steps of (a) culturing pluripotentstem cells in the presence of activin to induce differentiation intoendoderm lineages, (b) further culturing the cells of step (a) in thepresence of BMP-4 and bFGF and in the absence of serum to inducedifferentiation into a hepatic lineage, and (c) culturing the cells fromstep (b) with one or more cytokines that promote hepatocyte growth toproduce a population enriched for hepatocytes.
 69. The method of claim68 wherein activin is present at a concentration of about 30 ng/ml toabout 100 ng/ml, BMP-4 is present at a concentration of about 10 ng/mlto about 30 ng/ml, and bFGF is present at a concentration of about 1.0ng/ml to about 10 ng/ml. 70.-74. (canceled)
 75. The method of claim 68wherein the cytokines are selected from the group consisting of VEGF,human recombinant HGF, EGF, bFGF, and TGFalpha.
 76. The method of claim75 wherein VEGF is present at a concentration of about 10 ng/ml, humanrecombinant HGF is present at a concentration of about 20 ng/ml, EGF ispresent at a concentration of about 10 ng/ml, bFGF is present at aconcentration of about 10 ng/ml, and TGFalpha is present at aconcentration of about 20 ng/ml. 77.-80. (canceled)
 81. The method ofclaim 68 wherein the composition is administered by injection,implantation or infusion.